‘Chapman & Hall/CRC Mathematical at Comp AN INTRODUCTION TO SYsTEMS BIOLOGY DESIGN PRINCIPLES OF BIOLOGICAL CircurtsCHAPMAN & HALUCRC Mathematical and Computational Biology Series ims and scope hie sre sme cpu new development td sormrize wk ove wo spec of mater and compute ogy tid medi. asks o ena ‘hcp of emai ties! ed compatatonal mbar ino Boloy Oy DAIS ‘aca mpl sae escarsos an presen he aerate stitial emulations science, fondant ology sd bhnmpnerng. a well a ertiptiary "esate awoey te ld. The inlsion of cone examples and apps ad ‘ogeanrg txts leapt Mehl erred Series Bitors Dipwrnen Ses (inert f Oxford ai Ges Deptt of logy and Buna Balog nner of nae Senne Lesa ra of Mae mas thera Temes Mashonatial finte Unie of Ogun Poaomatis iv Cong Deparment of meal Bcc my psf the ere shold be abit of the sein ers veo ety (GRE Press, Tstor Francs Group Beas Ble Co eae SWIS 2NU Published Titles Cancer Modeting and Simulation Luigi Prezios Computational Biology: A Statistical Mechanics Perspective Ralf Blossey ‘Computational Jianfeng Feng Neuroscience: A Comprehensive Approach ‘Data Analysts Toots for DNA Microarrays Sorin Draghici Differential Equations and Mathematical Biology IS, Jones and B.D. Sleeman, Exactly Solvable Models of Biological Invasion Sergei V. Petrovsii and Lian Ba Lt ‘An Introduction to Systems Biology: Design Principle of Biological Circuits Uri Alon Knowledge Discovery in Peotcomics Igor Jurisica and Dennis Wighe Modeling and Simulation of Capsules and Biological Cells .Poscikiis Normal Mode Analysis: Theory and Applications to Biological and Chemical Systems Qiang Cui and Ive Bahar Stochastic Modelling for Systems Biology Darren J. Wilkinson The Ten Most Wanted Solute Anna Tramontano in Protein BioinformaticsChapman & Hall/CRC Mathensaiel ane! Computation AN INTRODUCTION TO Systems BIOLOGY DESIGN PRINCIPLES OF BIOLOGICAL CIRCUITS Uri ALON & Hall/CRCChapa aR rar ‘e200 by Tbr tani IE hapa ae asp fr Fac Group. rma sons stank Une utero Ameer on oper Ieuan sonnet ner 9743 sei 92 ce) TASSTESEALTScnm St aca acme sme pny the ao opatefhnbnk ny een eeu annie. ‘shtrtnans nov nen hve nventes nating 0 ‘Gorge on slo etn ween ean rom he pois. For pelt photocopy aw mati elton ram he rk, pape een economy ‘recog erst te Capri haste Caer ne {CC Feed ve Dans ASD, SeHTado LC mtx ntti a prosecestsend gation eu fer ore "icine testcase pasa ear yan essere eyes bem Trademark Notice Pet rer as oy tinea epee dems a ar as ony ‘Menno th ena abana ong saga Pabiewn ale ‘iecicwon antes dpi lp cea by Abn Ten Chipman bene mahal ecules!) fect apes rerencesip ania ‘enpentndbolgy. 2: Bpealapems Mathematics. TA IL Sees cqusn.2 A406 isthe Tne a Fans We ie For Paina and HananAcknowledgments 4 pleasure to thank my teachers. First my mother, Prina, who give much loving care to teaching me, among many things, math and physics throughout ay childhood, and ‘my father, Hanan, for husnor ané humanism, Ta my Ph.D. adviser Dov Shas, with his impeccable intuition, love of depth, and pedagogy, who offered, when I was confused about what subject to pursue after graduation, the unexpected suggestion of biology. To ‘ay second PhD. adviser, Davidl Mukamel, for teaching love of toy models and for the freedom to try to make a ess in the labs of iki Kamm and Yossi Yarden inthe biology building. To my postdoctors! adviser Sten Lesbler, who introduced me to the study of design principles in biology with caring, generosity, and many inspting ideas, To Mike Surette and Araie Levine for teaching love of experimental biology and for answers 10 almost every question. And to my other first teachers of biology, Michael Elowite, Eidad ‘Toahor, aud Tal Raveh, who provided unforgettable fist experiences of such things as centrifuge and plpete ‘An not ls have I learned from my wonderful students, much of whose research 4s described in this book: Ron Milo, Shai Shen-Ore, Shalev Mekovite, Nadav Kashian, Shmoolk Mangan, Etez Dekel, Guy Shin, Shiraz Kas, Alon Zaslaver, Alex Sigal, Nit 2am Rosenfeld Michal Ronen, Naama Geva, Galt Lahay, Adi Natan, Reuven Levitt and thers. Thanks also to many of the students in the course “Introduction to Systems Biol ©8y.” upon which this book is based, atthe Weizmana Institute fom 2000 t0 2006, for questions and suggestions. And special thanks to Naaina Baska for friendship, inspira tion, and for developing and teaching the lectures that make up Chapter 8 and patt of Chapter 7, To my fiends for much laughter mixed with wisdom, Michael Elowite, Tevi Tlusty, Yovalal Liso, Sharon Bar-29, Tal Raveh, and Ak and Uri Moran. To Euna and Osi, Dani aud ffeptibah, Niland Gidi with love, fo Glia Mocan with love For reading and commenting on all or parts of the muanusssip thauhs bu Dani Alo, Toei Tlusty, Michael Flowitz, Ron Milo, Shaler Kekovite, Haninah Margalit, and Arie (Cohen. To Shakey Iizkovtzfor devoted help wth the ectures and book, and te Adi Natan, foc helping with the cover design. ‘To the Weismann Insite, and especially o Benny Geiger, Veda Rotter, and Haim Harari and many others, for keeping out insittea place to payContents Quusmca 1 Introduction 1 Guru? = Transcription Neworks: Basle Concopis_ 2.1 Introduction 2.2 The Cognitive Problem of the Cell 2.3 Elements of Transcription Networks ? 2.2.1 Separation of Timescales 9 2.3.2 The Signs on the Edges: Activators and Repressors 2 2.3.3 The Numbers on the Edges: The Input Function 3 2.34 Logic Input Functions: A Simple Framework for Understanding Network Dynamics 5 2.3.5 Mult-Dimensional Input Functions Govern Genes with Several Inputs 6 2.346 Interim Summary 18 24 Dynamics and Respanse Time of Simple Gene Regulation 18 2.4.1 The Response Time of Stable Protein ls One Cell Generation a Further Reading 2 Exercises 2 Guyeng 3. Autorogulstion: A Network Motif 27 53.1 Ineoduction 2 3.2 Patlers, Randomized Networks, and Network Motifs 2 3.2.1 Detecting Network Motif by Comparison to Randomized Networks 29 3.3 Autoregulation: A Network Motif 30 3.4 Negative Autoregulation Speeds the Response Time of Gene Circuits 31 3.5 Negative Autoregulation Promotes Robustness to Fluctuations in Production Rate 3a 3.51 Positive Autoregulation Slows Responses and Can Lead to BiStabilly 37ali CONTENTS CONTENTS 4 Sum ” Gusrtoe 5 @ Temporal Programs and the Cabal Structure of Farther Reading 3 ——_Tanseription Networks 25 Exercises 38 5 Invoducton 75 5.2 The Singletnput Module SIM) Network Motif % Guaruie 4 a The Feedforward Loop Network Motif AT. 5.3 SIMs Can Generate Temporal Expression Programs. 7 441 nvoduction a 54 Topological Generalizations of Network Motifs 8 4.2 The Number of Appearances of a Subgraph in Random 5.5 The Muli-Output FFL Can Generate FIFO Temporal Order 8 Networks 2 5.3.1 The Mul-Output FIL Can Also Act a8 a Persisience Detector 4.3 The Feed-Forward Loop i a Network Motif 4“ for fach Ourpus o7 44 The Structure of he Feed Forward Loop Gene Cicuit “ 5.6 Signal Integration and Combinatorial Control Bi-fans 4.5 Dynamics ofthe Coherent Type-t FFL with AND Logic: “0 and Dense Overlapping Regulons 88 ‘Ab The C1-FFL sa Sign-Sensitive Delay Element 50 5.7 Network Motifs and the Global Sircture {46,1 Delay Following an ON Step ofS, I (of Sensory Transerption Networks 89 446.2 No Delay Following an OFF Step ofS, 52 Further Read ng 2 446.3 The C1-FFL Isa Sign Sensitive Dey Element 52 verses 2 ‘46.4 Sign Sensitive Delay Can Protect against Brief Input Fluctuations 52 Charts 6 m Network Motifs in Developmental, Signal Transduction, 46.5 Sign Sensive Delay in the Arabinose Sytem of E.coli sa and Neural Netwasks Pa 46.6 The OR Gate C1-FL I a Sign Sensitive Delay for OFF Steps ofS, 56 @linwducion = 4.6.7 interin Summary 56 6.2 Network Motifs in Developmental Transcription Networks 98 47 The Incoherent Types) FFL 37 6.2.1 Two-Norle Positive Feedback Loops for Decision Making 99 4.71 The Structure of the Incoherent FFL 7 6.2.2 Regulating Feedback and Regulated Feedback i 472 Dynamics of he H-FFL: A Pulse Generator 58 6.23 Long Tanerption Cascades ane Developmental ining 102 4.73 The I1-FFL Speeds the Response Time a 6.24 Interlocked Feed-Forward Loops in the B. subtilis Sporuation 4.74 Response Acceleration Is Sign Sensitive 6 Network 01 4.75 Experimental Stay ofthe Dynamics ofan 1-FFL 63 6.3 Network Motifs in Signal Transduction Networks 104 4.76 The Ways to Speed Your Responses tAntnerty Summary oa 64 information Procesing Using Multi-Layer Perceptions 106 4.8 Why Are Some FFL Types Rare? os ‘6.4.1 Toy Model for Protein Kinase Perceptrans 106 4.8.1 Steady State Logic ofthe I-FFL:, Can Tun on High Expression 65 oh.2 Muli-Layes Pereepons Can Perform Detailed Computations i 48.2 HFFA, a Rarely Selected Circuit, Has Reduced Functionality 65 6.5 Composite Network Motif: Negative Feedback and Oscillator Matis 115 4.9 Convergent Evolution of FFLS 68 6.6 Network Motifs in the Neuronal Network of C elegans ne 4.10 Summary 69 6.6.1 The Multi-nput FEL in Neurenal Networks 122 Further Reading 70 6.6.2 Multi Layer Perceptrons in the C. elegans Neuronal Network 5, Brerises a 6.7 Summary vw Further Reading 128 Exercises 129xiv a CONTENTS CONTENTS # ow Charis 7 Robustness of Potin Cie: The ample of 9.31 equim Bing Cannot Expin he Low Eo Rate Dar Cheol 5 ol nmune Reogrton vas 7A The Robustness nile bs 93.2 Kinetic Pooeating Inceass Fly of Cal Recognition 185 72 Bacterial Chemo of How Baca Think wae 5 Kinec Poofating May Occur in Diverse Recognition Pocenes 721 Chota Behavior, 136 inthe Ga 167 722 Response and Brat Adatation wy Fane Reading vee 7.3 The Chemotaxis Protein Circuit of E. coli 140 Exercises ‘188, 7.41 Alvactats Lowe th Activity fX i Cur 10 Ootinal Gane Cnet Dosen wes 702 Ant DeSean; Sn puma! Gare Creat Design —_ig3_ 1A wohl in Ei Aber: abt ad Foti ed 102 Opal spn tn aa Pet oe es 7411 Fine-Taned Model 13 10.21 The Benefit ofthe LacZ Protein 195 742 The Btka-Lcle Robust Mechanism for Bret Adypiston 124 The nul he Le? Pe a 743. Aten sig edb 102.3 The Fes onto a te Optimal Epson Level i Steady Sut Atty and Adaption Ties Ate an Tat v9 1024 Lseraoy Elation Expert some That Cl Resch v9 15 Inala Rousvess in Baer Chemotans v9 os keinewnaleteaive eee Funke Rad 151 Tepes or Freteises 132 10.4 Environmental Selection ofthe Feed-Forward Loop Cnet Robust Patering in Development 59 sue oO 1 nweetion 189 tomo ogoge le He Rb ‘a ra ta we 8.3 ceased Robustness by Se tance Mexphogen Deqfadsion 183 6.4 Network Mos Tha rove Deaton Feeback for Rous Patri 198 Curr 11 Demand Rules or Gene Regulation 25 8.5 The Robustness inpe Can Disingush between Mecha of 114 Invodicion 25 Feu Fly Patterning 166 1.2 The Savageau Demand Rute a7 Further Reading, wa 11.2.1 Evidence for the Demand Rule in £, colt 27 Exercises ma 11.2.2 Mutational Explanation of the Demand Rule 219 chars 9 Kinetic Potent vs 112.3 The rblem wth Meant Selection Arguments 20 St awd eR 113 Rules or Gene gultion Based on Minimal Er Loed 20 4.2 Kee ruieatng ol he Cent Code Can Reduce Er as 114 Te Section ese for Opin Reuon 2 si Molecda Reespion v6 113 Demand ules for Mult Regulator Systems 2 9.2.1 Equilibrium Binding Cannot Explain the Precision of Translation W 11.6 Summary 28 9.2.2 Kinetic Proofeeading Can Dramatically Reduce the Ertor Rete 180 9.3 Recognizing Sof and Non Self by the Immine System 12Further Reading 230 Evercices 230 Civ, 12 m Epilogue: Simplicity in Biology 23 Arsanis A # The Input Function of a Gene: Michaelis-Menten and Hill Equations 241 AL Binding of a Repressor to a Promoter 2a ‘A2 Binding of a Repressor Protein to an Inducer: Michaelis-Menten Equation 244 A Cooperativity of Inducer Binding and the Hil Equation 5 AA The Monod, Changeux, and Wymann Model 27 A. The Input Function ofa Gene Regulated by a Repressor 27 A. Binding of an Activator ta ts DNA Site 248 4.6.1 Comparison of Dynamics with Logic and Hill Input Functions 250. A. Michaelis-Menten Enzyine Kineties 250 Further Reading 251 Exercises 251 ‘Avtxbo Ba Muli-Dimenstonal Input Functions 253 BJ Input Function That Intogrates an Activator ane a Repressor 253 Exercises 255 Avinsian € a Graph Properties of Transcription Networks 257 (C. Transcrption Networks Ave Sparse 257 C.2 Transcription Networks Have Long-Tailed Output Degree Sequences and Compact input Degree Sequences 287 (C3 Clustering Coefficients of Tanscription Networks 259 C4 Quantitative Measures of Network Modularity 259 Alininy D a Coll-Cell Variability in Gene Expression, 261 Further Reading 264 Guossiay 265, Busieceary Dh Introduction ee When [fist red a biology textbook, it was like reading a thriller, Every page brought & new shock. As physicist, was used to studying matter that obeys precise mathematical Taw hut cells are matter that dances. Suctares spontancously assemble, perform eabo- Talc biochemical Functions, and vanish effortlessly when their wark is done. Molecules encode and process information virtually without errors, despite the fact that they are under strong thermal noise and embeded in dense molecular soup. How could this he? ‘Are there spevial laws of natare that apply to biological systems that ean help us to under- stand why they ae so different feom noting matter? We yearn for laws of nature and simplifying principles, but biology Is astoundingly lex. Fyery biochemical interaction is exquisitely crafted, and cells contain networks ‘of thousands of sich interactions, These networks ace the resi af evolution, which works by making eandom changes and selecting the oxganisnis that survive. Therefore, the structures found by evolution ate, to same degree, dependent on historical chance and are laden with biochemicat detail that requices special desertion in every case Despite this complexity, scientists have atempted to discera generalizable principles throughout the history of biology. ‘the search for these principles is ongoing and far from complete ts made possible by advances in experimental technology that provide etated and comprehensive information about networks of blogic interactions. Such studies he to the discovery tha one can, infact, format general avs that apply to biological networks. Because it has evolved to perforin functions, biological cicculty Is far from random or haphazard, It has defined style the style of systems that must fune- tuon, although evolution works by random tinkering, it converges again and again onto a defined set of circuit elements that abey general desig principles. ‘The goal ofthis book is to highlight some ofthe design principles of biological sys tems, and to provide a mathematical framework in which these principles can be used t0 understand biological networks. The main message is that biological systems contain an inherent simplicity. Although cell evelved to function and did not evolve to be compre- hhensbl, simplifying principles make biological design understandable to ws.2m CHAPTER This books ertten for students who have had a bie course én mathematics, Specialist ud gene naones are avoided, although detailed descriptions of several wel studied biological systems are peesented in order to demonstrate key principles. This hook pres- cents one path into systems biology based on mathematical principles, wih less emphasis ‘om experimental technology. The examples are those most familiar to the author. Other lirctions can be found inthe sources listed atthe end of this chapter, andin the extended, bibliography at the end ofthis book. “The aim of the mathematical mods in the book isnot to precisely reproduce experi ‘mental date but rather to allow intitive understanding of general principles. This is the art of “toy models” in physics: the belief that a few simple equations can capture some ‘essence of a natural phenomenon, the mathematical descriptions inthe book are there fore simplified, so that each can be solved on the blackboard oon a small pce of paper. ‘We wil see that t can be very useful ro ask, "Why is the system designed in such a way?” and to ry to answer with simplified made, ‘We conclide this introduction with an overview of the chapters. the fist part of the book deals with transcription regulation networks. Elements of networks and their dynamics are deseribed. We will sce that these networks are made of repeating occur reaces of simple patierns called network metifs. Each network motif performs a defined information processing function within the network, These building black clecults were ‘rediscovered by evolution again and again in different systems. Network motifs in her biological networks, including signal ransduction and neuronal networks, are also dis ‘cussed. The main points that biological systems show an inherent simplicity by employ ing and combining a ater stall set of Basle bailing block cieuits, cach for specie ‘computational tasks. “The second part of the book focuses on the principle of robustness: ological circuits are designed o that their essential function is insensitive to the naturally occurring flac- tuations inthe components ofthe ceeot. Whereas many circuit designs cam perform a ‘sven Function on paper, we will se that very few can work robustly in tk cll These few robust circuit designs are nongenerie and particular, and are ofienaesthstially pleasing. We will use the robustness principle lo understand the detailed desigr of well studied systems, including bacterial chemotaxis and patterning in uit tly develcpment ‘Te final chaprers describe how constrained evolionay optimization can be wsed to understand optial circuit design, and how Kinetic proofreading can sninimize errors made in biological information processing These features of biological systems reuse ofa small set of network motifs, robustness to component tolerances, and constrained optimal design, are aso found ina completely liferent context: systems designed by human engineers. Biological systems have addi tional features in common with engineered systems, suchas modularity and hierarchical design. These similritics hint ata deeper theory that can unify our understanding of evolved and designed systems. ‘This sit forthe introduction. A glossary of terms is provided at the cid of the book, and some ofthe solved exeseses aller each chapter provide more dell an topics dis ‘used inthe ssain text. I wish you enjoyable reading, INTRODUCTION #3 FURTHER READING iC Marland E, Wagner I. and Tyson [ (2005. Computational Cell logy, Springer. Fel D, (1996) Understanding the Control of Metabolm, Portland res. Heinrich, Rand Schuster, 8. (1996). The Regulation of Cellar Syston, Kluwer Academic Publiehers lipp E. Herwig, R, Kowald, A, Wing, Cand Lehrach, H, (205), Systems Biology in Prac ice: Concepts, Implementation and Application, Wiley XKriete, Aan il R. (2005), Corspuatonal Syetere Biology, Academic Press. Palson, BO. (2006) Systems Biology: Properties of Recontraced Networks. Cambridge Univer: sty Peas Sovageay. M.A (1976). Biockemical ystems Analyse A Stuy of Function and Design in Molecular ology. Addinon Wesleycunprer 2 Transcription Networks: Basic Concepts — 24 INTROGUCTION - “The cell isan integrated device made of several thousand types of interacting proteins Fach protein isa nanometer-size molecular machine that carries ota specific task with ‘exquisite precision, For example, the micron-long bacterium Escherichia coli isa cll that contains afew million proteins, of about 4000 diferent types (typial numbers, lengths, and timescales can be fond in Table 2), Gels encounter different situations that require diferent proteins, For example, when ‘sugar is sensed, the cll begins to produce proteins that can transport the sgor into the call and utilize i. When damaged, the cell produces repaie proteins. The cell therefore continuously monitors its environment and calculates the arpount at which each type of| protein is needed, Ths Information-processing function, which determines the rate of Production of each protein, is largely carried out by transcription networks. ‘Tae first few chapters in this book will discuss transcription networks, The preset chapter defines the elements of transcription networks and examines their dynamics, THE COGNITIVE PROBLEM OF THE CELL _. alls Hive in a complex environment and ean sense many diferent signals, including Physical parameters such as temperature and osmotic pressure, biological signaling mol ‘cules from other cell, beneficial nutrients, and harmful chemicals. Information about the internal state ofthe cll, such asthe level of hey metabolites and internal damage Ce, ddatnage to DNA, membrane, or proteins), isalso important. Cell respond to these signals by producing appropriate proteins that act upon the internal or external environment,6 = curren 2 [NE 2. Typical Panta Vc the ice cu al the Single Cold Ear Sucehaoyees cove (Yen Manin Cel onan Fist Peery Eek Yen) Minne ti) Taiwan 9 “aaa Prec aie nae sine Manone Sam Saco ae seiee Laity step eogens fangs aes Suet older idgate -10hp “ote “ip tt tty “inotip “ipo tp oo Zreake Sine it oh of ane proteinell m um Sitonontine tpn “Ose som one ach De topmne Diaioinctamil = tome te tcdescencdl | Deoouie Tetotinuitesgem Imi nin -20.ndadng ebpes ite pose Uineotandieapiin nin stni0 oan dnding ‘onine si cre Tetnmvaniaine Sn “onintoonr}h tonin tore i0h CAsreiontine 0a lichen) 2h hem) 20h noting wurenitow eure some ae “ ano baneon se ope tect ice sine wine tse sultnindngel (Luby) (aM ny) oan Sat mac peace tru) eases aise anserten tor angio DNA ste Muon te a0 cio" 10th lectin igenertion Ty ase ple DA Ter TRANSCRIPTION NETWORKS: BASIC CONCEPTS a 7 “To represent these envieonmental states, the cll uses special proteln called transcription factors as symbols, Tronseription factors are usually designed to transit rapidly between active and inactive moleculte stats, at @ rate thet is modulated by a specific envivon ‘ental signal np). Each ative transcription factor can bind the DNA to regulate the rate at which specific target genes are read (Figure 2.1). The genes are sea (transcribed) into mRNA, which is then translated into protein, which cam act on the envitoninent “The activites ofthe transcription factors ina cell therefore ean be considered an feral sepresentation ofthe environment, For example, th bacterium Eco has an internal rep: ‘resentation with about 300 degres of fecedoe (transcription factors) These regulate the ats of production off coli’ 4000 proteins. “The internal representation by set of transeription factors isa very compact desi tion of the mysiad factors inthe environment, Ht seems that evolution selected intemal representations that symbolize states that are most important for cell survival andl grow. Many different situations are summarized by a patticuba transcription factor activity that signifies "Iam starving” Many other situations are sunimatized by a different ta scription factor activity that signifies "My DNA is damoged” These transcription factors, regulate their target goncs to mobilize the appropriate protein responses in each cas, 2.3 TLEMENTS OF TRANSCRIPTION NETWORKS “The interaction between transcription factors and genes is described by transcription net works. Let us begin by broly describing the elements of the network: scription factors, Each gene ia stretch of DNA whose sequence encodes the information il GRR 21 The mapping hewn sions sia toner floes the ell athe enesthat hey rele De cntonnenal pal atte specie aseipon fo utens. Theta Scion factors, hen ate bind DNA che the assent af peg eae te ate a Uihich mRNA rode he RUA hen tals io pon ence, assign for ese the rte at which the pin ncoed bythe ene are peed Tes poeins atthe env toner livernt esti Sone potens re Cente ans actors tat can ata ees theinceded for pruduction ofa preten. ‘Transcription of a gene isthe prucess by which RNA polymerase (RNAP) produces mRNA that corresponds to that gene's coding sequence The mRNA is then trnsfted into a protein, also called the gene product (Figure 2.28) ‘he fate at which the gene is transcribed, the number of mRNA produced per uni time, is controled by che promoter, a ogultory region of DNA that precedes the gene (igure 2.20). RNAp binds a defined site (a specific DNA sequence) atthe promoter (Fg. lire 2.23). The quality af this site specifies the transcription rate of the gene! Wheres RNAp acts on virtually all of the sille genes ate due to transcr nes, changes in the expression of spe= on factors. Each transcription factor modulates the tran rat of a set of target genes, Transcription factors affect the transcription rte by binding specific sites in the promoter ofthe regulated genes (Figure 2.2b and o). When ‘bound. they change the probability per unit time that RNAp binds the promoter and pro: «duces an mRNA molecule The transcription fsetors thus affect the rate at which RNA initiates anscription ofthe gene, Transcription factors cam act as activators that increase the transcription cate ofa gene, oF a8 repressors tht reduce the transcription rate (Figute 22band 0, ‘Transcription factor proteins are themselves encoded by genes, which ere regulated by ‘other transcription factors, which in turn may be regulated by yet other transcription fac~ tors, and so on. This st of interactions forms a transcription network (Figure 2.3) The transcription network describes all of the regulatory transcription interactions in a cell {or atlest those that are known. In the nerwork, the nodes are genes and edges represent \canseriprional regulation of one gene by the protein product of another gene. A directed ‘edge X >> ¥ means thatthe product of gene X isa transcription factor protei that binds the promoter of gene to coateol the rate at which gene ¥ is transcribed, ‘The inputs to the network are signals that carry information from the environinent Fach signal isa small molecule, protein modification, of molecular partner that dicectly aifects the activity of one of the transcription factors, Often, external stimuli activate bio, chemical signal transduction pathways that calminste in a chemical modification af spe- «fe transcription factors, tn ather systems the signal cen bes simple asa sugae molecule "hat enters the clis and dicectly binds the transcription factor. The signals usually cause « physics] change in the shape ofthe éranscription factor protein, causing i to assume an setive molecular state. Thus, signal S, can cause X to rapidly shift to its active state X* Vind the promoter of gene Y, and increase the rate of transcription, leading to increased production of protein ¥ (Figute 2.2. ‘he network thus repeesents a dynamical system: after an input signal arrives, vans scription factor s change, leading to changes in the production tate of proteins. Some ofthe proteins are transcription factors that activate additional genes, and so on. script Sie dermis tcl at of RNAP tee "ether stcan amt am pc conten nce BRAD ‘ats tenon ce Cand aracierone nA ware ‘eine pid preva Tie 2, Paes tr che Faby pe ne TRANSCRIPTION NETWORKS: ASIC CONCEPTS 9) xa eo I oats ropes NN Pf remeron ® t Goer a) Fach gee uu pcedd bys sepa hv yecii ste(DNA sequence that en ind RNA plynerise (RNA, comple of seve peti that mean eye thea yateste MINA ‘hat coreg tote gne coding sequence. The proces frig then RNA neal scion Te "DANA is he ral ino prin (9) An achat. Xi transcrip fat prot tht ceases ‘heat miRNA tosrption when t binds the promote The activo typ tasks ll betoe ctv ard native fms Init civ form thas high aly wo spel eo ses) on he ae, The signal Sy ness the eobablty tha Xs nits active aXe Xba piste nthe pres Df gene ¥oeceae ranserpion ad eodectan of poten) A epson © wa amp tee protein tht decrease the rate of MRNA tase pton when Wd he romeo, The sah Sere "he poaiy that Xin as active form, X"X- bie asp ein ie promoter a gee Yt deca ‘easerinion and pradztion of potin F. The eest ofthe preteins are not transcription factors, but rather carryout the diverse Fune- ‘ions of the living cells, such as building structures and eatalyzing reactions, 23.4 Separation of Timeseates ‘Transcription networks are designed with a strong separation of timescales: the input signals usually change transcription factor activities an a subssecond timescale. Bindingto CHAMTER 2 ot eee ay * ‘ or Notronsetiption t seul ar ® inter Og ae © CURE 22 eomtinsed) een a iv ICURE 2.3 & tanseription network that reptesets aout 20% af he transcription teractions inthe tem & de Ne sre gens lr groups Of gees coded on the tme MRNA ald operon) AN edge ¥ deci ap amet tes sn ‘Xd non ¥ (Chip 6) Inman ener elton wba nee gly eacdoa ‘ating sips hte i of mcg syne Ths eine Ean Pa ae no he ieee pet mucins wh con everett ay ee TRANSCRIPTION NETWORKS: HASIC CONCEPTS 13 23.3 The Numbers on the Fdges: The Input Function ‘The edges not only have signs, but also can be thought to carty umbers that correspond to the sitength of the interaction. The strength of the efiect of a transcription faetor on the transcription cate ofits target gene is described by an input function Let us conser fist the production rate of protein Y contrlled by a single transcription fector X. When X regulates ¥, represented in the network by X ¥, the numberof molecules of protein ¥ Produced per unit tint isa function ofthe concentration of Xin its active form, X tof prolction of ¥ = 0X") ea ‘Typically, the inp function ((X*) is « monotonic, S-shaped funtion. It isan increas- ing Fustion when Xis an activator and a decreasing Function when X i repressor (Fg ‘oe 24). useful funtion that describes many real gene input Functions is clled the Hill function. "The Hill function can be derived from considering the equilibri binding of the transcription factor to itssite on the promoter (ce Appendix A for further details) ‘The Hill Input function for an activator ta curve that rises fom zero and appronches a ‘maximal saturated evel (Figure 243) Hl fonction for activator 23.2) ‘The Hill function has three parameters, K,B, and n, The fist parameter Kis termed the activation coefficient, and has units of eoncenteatoa, I lefines the concentration of sctiveX needed 10 significantly activate expression, From the equation it iseasy tose thet balfmaxioal expression is ached when X* = K (Figure 24), Ihe value of Kis elated to the chemical affinity aetween X and its ste on the promoter, a wel as atone! factors. “The second. perameter in the input function isthe maximal expression level of the promoter, . Maximal expression is scached at high activator concentrations. X* >> K, ‘because at high concentrations, X* binds the promoter with high probability and stime lates RNAp to prodiuce many mRNAs per unit tie. Finally, the Hill coefficient n governs ‘the steepness ofthe input function. The larger isn, the more step-ike the input function (Figure 240). Typical, input funetione ate moderately steep with n= 14 AAs do mony functions in biology, the Hill function approaches iting value a high levels of X, rather then inereasing indefinitely. Ths saturation of the Hil netion at high 2X concentration i fundamentally due to the fact that the probability thatthe activator binds the promoter cannot exceed I,no matter how high the concentration of X*. The Hill ‘equation often describes empirical data with good precision. For a repressor, the Hill input function isa decreasing S-shaped curve, whose shape depends on thee similar parameters foey 2 B 4 Hi input fanction for vepresior (2.3.3)= cuapres 2 pci ¥ peasy genni 7 » Hciet 2.4) tap octet X esrb by Hota wih coef a= 9,2 ad ‘ tromoter acs pte ea factn te cee oF Xin ete fra) Alea shew ‘hep unin, ae calle eg opt oacton The anil pom civy yaad ete treba Foran a arg gee he Cocenation CX need or 3 asl statin () ep fae hms spresiorX decd by Hl orton wth ll coca and lve town the co ‘espording lag npt fonction ep Fncton. The mana vwepeased omer actly and Ks thet or ecreion as age ee Ihe coentatin af needed for 50% maul epee TRANSCRIPTION NETWORKS: BASIC CONCEPTS @ 15 Since sepresor allows strong transcription af gene only when ts 301 Bound othe promoter, this function can be desved by consiering the probability thatthe promoter {sunbound by X* ce Appendix A). The maximal production rte Bis obtained when the repressor docs nt bind the promoter at sl (igure 2.2) that when X°= 0, HalEmaxt ‘nal repression i eached when the epeessoe activity seal to K, the gee’ represion ovfficent. The Hil coeficen » determines the seepnes of the input funtion Figure 24). Hence, each edge in the network can be thought to cany atleast thee nuesbers B, ,and n, These numbers can eeadily be ted during evolution. For example, K can be hanged by mutations tht ater the DNA sequence ofthe binding site of Xin the pro- Inte uf gone, Essig fw ingle DNA lets Udng ite wae ang ct weaken the chemical bonds between X and the DNA and change K. The parameter K ca also be varied if the postion of the binding ste is changed, aswell as By changes in sequence ouside af the binding site he ater ets are curently nt fly understod}. ‘Similely, the maxima activity Bean be tuned by mitations in the RNAp binding ste or ‘many other factors. Laboratory evolution expetiments show that when placed ina new ‘vironment bacterin can accurately tune these numbers within several hundred genera- tions to reac optimal enresion level (Chapter 10), In other wor, these numbers are ‘under slecton pressured can heritaly changeover many generations environments change “The input functions we have described range from 2 transcription rate of 610 10 a saimaltanscrition rte. Many genes have «nonzero minimal expression level. This ic called the goer basal expression level. 4 basel evel canbe described by adding tthe input function a ter By 23.4 Logie pat Functions: A Simple Framework for Understanding [Network Dynamics Hill input functions are useful for detailed modes. For mathematical clarity, however, it Is often useful to use even simpler functions that capture the essential behavior of these Input functions. The essence of input functions is transition between low and high values, with a chasacteristc threshold K, tn the coming chapters, we will often appreximate inp fonctions in transcription networks using the logie approximation (Figure 24) and Kaufman, 1975 Thiefry and Thomas, 1998). In this approximation, the gene is either OFF, (2%) ~ 0, oF maximally ON, s[X*) =f. The threshold for activation is K. Hence logic Input functions are step-like approximations fr the smoother Hill functions, Por ativa- tors, the logic input fanction can be described using a step-function 8 that makes a step Jwhen X* exceeds the threshold K: 00) =BOQK*> K) epic approximation orautvator 234) where @ is equal to O or 1 according to the logic statement in the parentheses The logic approximation is equivalent to a very steep tll function with Bill coeticient 9% (gute 24a),16 @ CHAPTER 2 Similarly. fo repressors, a deereasing step Function is appropriate: (00) BOO KOC > K)~ xe ANDY e326) For other genes, binding of either activator sulicint. This resembles an OR gate 12,17) = BOO > KORY > K)~ ORY? ean Not all genes have Boolean-like input Functions. For example, some genes display a SUM input function in which the inputs are additive (Kar and Alon, 2004) FOG.) = BX EBL" 038) Other fanctions are also possible. For example, a function with several plateaus and Uhresholds was found the lac system of E. colt (Figure 2.5) (Se color insert flowing bp 112). Genes ns mu-cllolar organisms often ceplay input functions that can eae late elaborate functions of dozen oF more inputs (Yuh et al, 1998; Davidson etal, 2002; Beer and Tavazoie, 2004) ‘The functional form of input functions can he readily changed by means of mutations Inthe promoter of the regulated yene. For example, the lac input fenction of Figuee 2.5 «an be changed to resemble pure AND of OR gates witha few mutations i the lac pro ‘moter (Mayo et, 2006). It appeais thatthe precise frm of the input function of each saci under selection pressure during evolution, TRANSCRIPTION NETWORKS: BASIC CONCEPTS @ 17 i ” 4 2 ap lonete ToL ay eon’ aston FIGURE 25 (See er inert fawing page 112) Tw deesiaal np orton) ng ention ‘weasel inthe lee ponte of Fa at anon of mp sale ines cAMP ad TS yan ANDike inp uncon, nich shows igh promo aay any both np ae ese) An ‘OR inpat nto ht shows high promoters ete snl Is ese (om Stet 3008)vam CHAPTER? ‘terns Summary “Transcription netvorks deseribe the tanscripton regulation of genes, Each node repre: Scns a yene. Edges denoted X + Y mean that gene X encodes for a transcription fact protein that binds the promoter of gene Y and mvodulates its rate of tanseription. Thus, the pratsin encoded by gene X changes the rate of production ofthe protin encoded by ign ¥. Protein , in tar, might be a transcription factor that changes therate of produc ‘om of 2, and 0 of, forming sn interaction network. Most nodes in the network sta for ‘genes that encode proteins that are not transcription factors. These proteins carry out the aris functions ofthe cll “The inputs to the actwork are signals that carry information from the environment and change the activity of specific transcription factors, The ative transcription factors bind specific DNA sites in the promoters of thelr tar: ‘get genes to control the rate of transcription. This is quantitatively described by input fonctions the cate of production of gene product ¥ is 2 function ofthe concentration ‘of active transcription factor X*. Genes regulated by multiple transcription fectors have ult dimensions! input factions. ‘The input functionsare often rather sharp and can be ‘spproximated by Hill functions o logic gates. very edge and input function is under selection pressure. A nonuseful edge would ‘rapidly be lst by mutations. I only takes a change of one ofa few DNA letters in the binding site af Xin the promoter of Y to abolish the edge X + ¥ Now we tara tothe dynamics of the network. 24__ DYNAMICS AND RESPO! lot us focus on the dynamics ofa single edge im the network Conside a gene that is ted by a single regulatos, with no additional inputs (or with all other inputs and posttranseriptional modes of regulation held constant over tine! Ths transcription ‘interaction is deseibed in the network by xo ‘wich resus “transcription factor Xeeyulates gene ¥." Once X becomes setivated by 2 sig ral, ¥ concentration begins to change. Let us calculate the dywamies of the concenteation ‘ofthe gene prot, the protein ¥ and is response time, Inthe absence of its input signal, X is inactive and Y is not produced (Figure 2.2). ‘When the signal S, appeats, X rapidly transis to its active form X* and binds the pro- mer of gee ¥. Gene Y begins t be transcribed, and the mRNA Is tandate, resulting Tintacwia en rly an oer atone erg hat ernst a hear aN ‘nedpet Yani ef he gen pean Kener ancy thelr aero ly regan avery pe ht yet ces aang he wing par eof dps he NA, rte san coe ime oy tone ay mtg pry peg KAA ih gicaysn seat heel Masyhe of gelaonae pe TRANSCRIPTION NETWORKS: ASIC CONCEPTS #19 ‘accumulation of protein ¥. The cell produces protein Y ata constant ate, which we will denote B (units of concentration per unit time) “The production of Y is blanced by two processes, protein degradation (its specific Aestruction by specialized proteins in the cll) and dotion (he reduction in concent tion due to the increase of cll wolume during growth). The degradation rate i Quy and the dition rate isa, giving 2 total degradationilation rate (in units of time) of = Oy 4 Oh ean “The change in the concentration of ¥ is due to the difference betwen its production sd degradationsilution, as described by a dynamic equation’ avidt=B-0¥ a2) [At steady state, Y reaches a constant concentration Vy The steady-state concentration can be found by saving for AV = 0. This shows thatthe steady-state concentration is the ratio of the production and degradation/iution rates Ya= Bla a3) “This makes sense the higher the production rate the higher the protein concentration reached, Y,. The higher the degradation/dilution rte a, the lower is Yq so that production of ¥ stops (B= = O isan exponential decay of ¥ concentration ‘What happens if we now take way the input ign 08 The selution of Equation 242 with (gare 2.69) W=Yeer" ean How fst docs ¥ decay? An important measure forthe speed at which Y levels change fs the response time, The response time, Tj, is generally defined as the time to reach Dralfway between che inital and final levels in a dynamic process. For the decay process ‘of Bqustion 2.44, the response time isthe time to reach halfway down from the initial level ¥, tothe final level, ¥ = 0. The response time, therefore, is given by solving forthe time when ¥() « Yq2, which, using Equation 2.4, shows an inverse dependence on the degradation/ilation rate a= Hoga eas) "hse hs en el see thee dys of esa doy lar xa Mon a 52, Iggimrcnaentapeemen wit hgb-seton dante eer ae une colon eva ana "i Sarin expos gre ctrl nee AR and A 280) Ne ht oe Prenton rough ath B= Fieyiermtant asberere the tine fc tomes a trio whe sia nego Lecas taltconpacd a thesoni ne eps eel Bam Take? 220m CHAPTER 2 o HUE 6 3) Decyof protein conentation ftring wake op proton rte The response ‘ethene aks the coca to reac Malt raion, Frys lagi Th epost oo he fou graphically by seine whe the earw roses he zl dashed ie ace faleaySeeaen ‘heap ed yt otf the ssc.) Rin “avai Typ gf tc ist poi Bride ing ancien he tie aes the dare ach half fs ctmlationsappesinaely cw tine, Note that the degradationilation rate a directly determines the response time: fast Alegradation/ditation allows rapid changes in concentration. The production rate affects the steady-state [evel but not the response tine, Some proteins show rapid degradation rates (large a). At steady-state, this lads to a scemuingly futile eye of production and destruction. To maintain a given steady state, = Bila, requires high production B to balance the high degeadation rate a."The benefit of such futile cycles fast response imes once change is needed, We have scen that loss of input signal leads to an exponential decay of Y. Let ws ‘ow consider the oppusite case, in which an anstisalated cel with Y = 0 is provided with a signal, so that protein ¥ begins to accumlte If unstimulated gene becomes TRANSCRIPTION NETWORKS: HASIC CONCEPTS w 21 suddenly stimulateé by a strong signal She dynamic equation, Equation 24.2, results in an approach to steady sate (Figore 2.60) Y=, 0-9 246) “The concentration of ¥ rises from zero and gradually converges on the steady sate Yq = Bla, Note that at sary times, when at 6, Can this be used to form a quasisteadystate assumption that mRNA levels ae at steady state with respect to slower processes? What isthe effective protein production rate B in terms of Bay @gs and p? What would be the response time ifthe mRNA lifetime were much longer than the protein lietiene? thea alactonidase in Echerihia cel as a anction af growth. Bacher ops Ac, Salons bas-ceb Rosenfeld, Wand Alon, U. 2009). Response delays and the steuctre of ransergion network. ‘Mol Bio, 329-688-654 EXERCISES 2 A change production rate A pine ¥ wi sine elation is proce ata con Stantrte yt rsction te suey Sit tient ete 4. Caleulate and plot the gene product concentration Yt) bs. What i the response time time to reach halfway between the steady states)? Solution (fr part a 1. Let us mak the time when the shift occurs as t = 0. Before the shift, ¥ reaches steady state at level Y(C= 0) = Y= Bula After the shi, d¥idt=By-a¥ (ery ‘The solution of such an equation sgenerally ¥ = C, + Ce, wher the constants , and C; need to be determined so that V(t 0)» fifa, and ¥ at long times reaches is new steady state, ia, This yells the following sum of an exponential anda constant: 1. The dynamic equation forthe concentration of mRNA of gene Y, Y, AY fat = By 8 Ya e239 ‘The dynamical equation for the protein product is du to production ofp copies permRNA and degradation/diluion at rate Yara wa) viet bb Inthe typical case that mRNA degradation i aster than the degradationéi tion ofthe protein product, we cam assume that Y, teaches steady slate quickly jn comparison to the protein levels. The reason i thatthe typieal time for the ‘mRNA to each steady state isthe response time log). which is much shorter than the protein respanse time loy(2/a because a, >> a, The steady-state mRNA level is found by setting dY t= 0 in Equation P23, yielding You = Balan (23) Using this for Vg in Equation P2.4 yields the following equation forthe prt production rate AYE =p Baldy @Y (29)24 @ CHAPTER 2 ‘In other words, the etlective protein prosacton rte, which isthe fest teem on ‘he right hand se of the equation, sequal tothe stesly-state NA level times {he umber of proteins transate from exch mRNA, B= PBuloy (727) 2.3, Fime-dependent production and decay. A gene Y with siezple regulation has atime- \lependent production rate (0 and a time-dependent degradation rate al). Solve Frits concentration 36 funetion of ime, Solution: Verify by taking the time derivative thatthe following is correct vw spf fat") &') 110) + [BC)exptfat”) A) a) (8) 24, Gaseles Cansider a eayeade of three activators, X-» Y > Z. Protein X is intially Preset im the cell in its inactive frem. The inp signal of X, Sy appears at time £50, Asa result, X rapidly becomes active and binds the promoter of gene ¥, 50 thot protein ¥ starts to be produced at rate , When Y level exceed a threshal Ky ‘gene 7 begins to be teanseribed. All proteins have the same degradationtilation Fite a. What is the concentration of protein Z as a function of time? What sits Fesponse tine with respect to the time of addition of $,? What about a easeade of thre repressors? Compare your solution to the experiments shown in Figure 27 2.5, Fan-out. Transcription factor X regulates two genes, ¥, and Yy, Draw the resus network, fermed a fan-out with two targct genes, The activation threshols for these ses are K, and K;, The activator X begins to be produced at tine t= Oat rate Bis signal i deprodediiluted at rate a, and it signal, is preseat throughout What are the times at which the gene product, the stable proteins Y, and Ya. reac halfway theie maximal expression? 2.6, Pulse of activation: Consider the cascade uf exstcise 24,"The input sgaalS, appeats at time 0 ora pulse of donation D, an shen vanishes (6) What te coneenralon YON? (6) What is the minimal pulse duration needed for the activation of gene 2? {@ Plt the maximal level reached by the protein Z a function of pulse duration D. TRANSCRIPTION NETWORKS: BASIC CONCEPTS 25, Cog TN tse wae SC Boe andsup 2 ne Cegratons o IGURE 27 (6 A ansptionl asad made of represen al The ansrpion cascade KY + iste of wo ells eepressor land TRB which aren ghee ton ates cal The csade was oig genetic npn. by cambnng ie appt mer DRA et tothe ppp gees Teh was det sho spe the rsh lors poem see sli A epriec fo the secon cascade step. Te bata thas orn een In pops 1 he poe tty ‘eplaed by TtR a pert asain urd tot ocprne at rps rare ent pita gene atnga epee fr the sland sep. (Responds ane eda ove cel ger on pe sce ste, Test step in the cascade enn ean to th ince HFT tha acne he "pres lac This nchation kad tothe prdctiona th repeisoe eR hat when pes tule mantis a deree in the acti of the scan tp promatr The experiment wa cated oust ‘organ which show dierent el gevertion es (the peneration te abet te flange 127°C camarel to 3), The an shor nen i othe cl pretation nei exch eons (rom Rast and An, 2003)charter 3 Autoregulation: A Network Motif 3.1 INTRODUCTION Jn the previous chapt red the basie dynamics of «single interaction in a (ran scription network. Now, let us take aJook at area, lve transcription network made of ‘many interaction edges (Figure 31), Asan example, we will use a network of transcription {ntrsetions of Escherichia coli that inckides about 20% ofthe organism’ genes (Shen-Ort etal, 2002), ‘This network looks very complex. Oe goal will be to define understandable patterns of 1 serve as building blocks ofthe network, Mealy, we would lke to under ofthe individual build stand the dynamics of the eatite network based on the dynam Ing blocks. I this chapter, we wil: 1, Define 2 way to detect building block patterns in complex networks, called net- work motifs. 2. Fxamine the simplest network motif in transcription networks, negative autoregulation, 3. Shove that this metif has wseflfanetions speeding up the response time of tran sctiption interactions and stabilizing them, ANDOMIZED NETWORKS, AND NETWORK MOTIE “The transcription network of F, coi contains numerous patterns of nodes and edges approach will be 1 look for meaningful patterns on the basis of statistical significance. ‘Todefine statistical significance, we compare the network to an ensemble of randomized networks. The randomized networks are networks with the same characteristics asthe seal network, (gs the sume number of nodes and edges as the real network), but where the connections between nodes and edges are made at random. Patterns that occur in the real network significantly more often than in randomized networks are called network otifs (Milo etal. 2002; Shen-Ore el, 2002) w atch Mea28 oe CHaAPIEK F nee te oilers in Fl Nes tha one spond wo gees ht ence unser pion hte poten hat el his vn promoter ee eplting sons repress late shonin Hack. Tc not comet in ihe ig chptrs hat bout N= 420 odes 50 els and y= 4D sce (9) Bsomple ofall ‘etworandHsrendomieed Enlor Rent wecsin, wih teams nie of moder and eee “The basic idea is that patterns that occur in the real network mich more often than in randomized networks must have been preserved over evoltamary timescales against mutations that randomly change edges. To apprecite this, note that edges are eal lost Ina transcription network, As previously mentioned, a mutation that changes single DDNA leter in a promoter can abolish binding of transcription factor and cause the loss of an edge in the network. AUTOREGULATION: 4 NETWORK MOTIF. @ 29 Such mutations can occur at a comparatively high rate, a can be appreciated by the fo lowing example. A single bacterium placed in 10 lof liquid nutrient, prows and divides ‘o reach a saturating population of about 10" cells within less than a day This population "therefore underwent 10 DIA replications. Since the maration sate is about 10 * pe let- fer per replication, the population will include for eseh lete ithe yenome, 10 different bacteria with a mason i that letter, Thus, a change of any DNA letter ean be reached ‘many times over very rapidly in bacterial populations. A similar rate of matations per {generation per genome oceuss in multi-cellulur organisms (for genome sizes and tnata- tion rates, sce Table 2.0, Similarly, new edges can be added to the networks by mutations that generate bind ing ste for transcrition factor X in the promoter region of gene ¥. Such sites can be Benerated, for example. by mutations or by events that dupliate or reposition pieces of 4 genome, or that insert into the genome pieces of DNA from other eels (Shapiro, 199). Hence, edges in network motifs must be constantly selected in order to survive randomiza- ion forces. ‘This suggests that if @ network motif appears in a network mach more often than in ‘randomized networks, it must have been selected based on same advantage t gives to the organism. Ifthe moti did nat ofer selective advantage, it would be washed out sad ‘occur about a aten sn randomized networks. 3.2.1 Detecting Network Motifs by Comparison to Randomized! Networks To detect network matifs, we need to compare the real network fo an ensemble of ran domized networks, We will frst consider the simplest ensemble of randomized networks, lntcoduced by Erdos and Renyi(Frdos and Reny, 1959; Bollobas, 1985). This makes cal K, a production staximal, namely, (8) = Prwhen Xs smaller than K. This was deserbed in Chapter 2.4 using the step function fo =poK lazion, The former shows response times, whereas the iter speeds response times. The slow dynatnies provided by positive autoregulation can be useful in processes that take relatively long time, suc as developmental processes (sce Chapter 6) Such slow processes «an benefit from prolonged delays betwees the production of proeis sesponsible for dif ferent sayes ofthe proces In addition, when the rate of postive autoregulation is strong compared to the deg radstioniiltion rte the eystem cat become bi-sable (exercise 3.4), Once the gene Is activated, it is lacked into astute of high expresion and keep iself ON, even fer the ‘origina activation Input has vanished (Cartier and Keasling, 1998; Demongeot ct al, 2000; Hecske etal, 2001; Fertll, 2002; and Isacs etal, 2003). This type of memory cle- ‘uit is used in developmental transcription networks to make iereversible decisions that lock a cel into a particular fate (eg to determine the typeof tzsue the cll wall become in a mult clita oxgunism; see Chapter 61. 26 SUMMARY - _ Negative storegulatbn isa network mia pttera tha recurs thoughout the aetwore atnumbers much higher than expected in random networks “To understand why negative auoregultian isa network mati we analyzed sts dypamic lechavior. The dymamve analysis can be peated asan engineering story Think of evostion 4 an engineer working to design a gee circuit hat would reich a dsiced steady-state oncentetion X,- Oe posible desig, design A, simple ceglation witha production tate st to reach X,, Design 1 negative aoreglation, witha atongcr nial prodve- tion rate, which, 8X bls up ir ppeseed to el inthe Seiad ted state “The second design ae the advantage hat the goa, X,, reached foster, Furthermore, the Auctuatons around X, cut variations in production rate we ele the second, ato- "egulted desig tn sn imaginary competition betwen two species deal excep that ‘oneuses ciel A athe second uses eu thelr would have asclective advantage Over evluionur Limes, strutores that have engineering vantages would tend 10 be ‘elected and appears ntoork sai FURTHER READING ‘Beste A. and Serauo, L200), Engineering tbily bn gene networks by autoregulation "Nature 405: 590-39Rosenfeld Bowie. MLB, and Alon, U. (2003). Ney time of teantersionastworks Mo Bal, 323 985-79. Savagent, M.A. (1976). Riochemical Systems Analysis A tudo Puction and Drag in Molecular ioogy. Addison. Wesley Chap Savagenw. M.A 1978. Comparison of cass! and aut genovs systems of reputation i tue ‘hl operons. Nature, 252 546-549, EXERCISES ‘that cooperatively represses ts own promoter (described! by a Hill function wit Hill coecient axe = Bl + OUKY) =a wan) How muuch faster is the response than in non-autoeegulsted circuits? Use the approximation of strong autorepresson, thats, (X/K)"2> Solution {nthe limit of strong autorepression, we can neglect the 1 in the derominator of the Jat function as soon as (X/K) >> 1, and we have AXIat= KIX aX, (a2 ‘Yo seve this equation, multiply both sides by X* and switch to the new variable, [Note that duflt= (n+ 1) X*dX/d. The equation now reads: duldt=(0 +) BKO-(oe aw (ra) ‘The solution of this linear equation is simple exponential convergence to steady sat, the same asin Chapter 2 ung deen ex ‘Switching back tothe original variable X, we have: Ke Xe) eteevanpenn es) The sesponse ae is Found by X(T) = Xq/2, This yes “hen ihcappronmation aa Rt tha ety at codigo ation P32.K, =X a melee tlio ht he Bn Sealy aeovcea eps intenireltetnatfntns A I arh H ie autoregulation speed te response 32, = {(0-+ Da)" logtaniae— (P36) “Te response time decreases with n, For n= 1,2, 3, the ratio of Ty 4 the response time of simply regulated genes 7," =loy(a) is about TT, = 0.2, (0.06, and 22, respectively. See Figure 3.5 for the dynamics of «strongly atorep= Inte gene with = 1. Te sharper the negative autoregulation (higher nthe more ‘the system approaches the sharp logie function Init discussed inthis chapter, and the faster t esponds Parameter sensitivity, Analyze the robustness of the steady state level of X wi respect to cell-cell variations in the production rate fh forthe system of problem 21 Calelate the parameter soaiity cooficiont (Savagrah, 1996: Goldbeter and ‘Koshland, 1981; Heinrich and Schuster, 1996) ofthe steady-state concentration with respect to B.'the parameter sensitivity coellcient of property A with respect to parameter B, denoted S(A, B), is defined asthe relative change in A fora given small relative change in B, thats, : S(A,B)= (GAWD) = (BIA) AAI (any Solution a3 ‘The steady-state levels found from Equation P32 using ANI = (Bra Ky 3a) yielding Xe “The parameter sensitivity, which describes relative changes in steady state due to changes in production rate i 80% eB = (BIKA) AX AB = Mn #1) 39) “Thus, senstivity decreases with Hil coefficient. The higher n isthe weaker the dependence ofthe steady state on B. In other words, robustness to variations in pro- ‘duction rales increases withthe Hill coefficient For Hill coefcient n = 4, for example, SCX, 8) = 1/5, which meons that 9 103% change in yields only 2% change in X,. In the limit of very high nthe steady- slate does not depend st all on prodiction of degradation rates, X= K. This isthe steady-state solution found in the main text for the logic input function. Simple re ‘ulation i equivalent 10m = 0, so that S(*,B) = 1. This means that a small change of 1 in production leads to the same change ofx% in steady-state. Atoregulated cascade, Gene X encodes a repressor that represses gene Y, which also encodes a repressor. Hoth X and negatively regulate their own promoters. ate starting from an inital con fs of X and Y? What ae the response 8. AV time 1 = 0, X begins to be produced at centration of X = 0, What are the dyn bw3A, 35, 36, times of X and Y2 Assume logic input functions, with eepressin thresholds Ky. K,, for the action of X on its own promoter and on the ¥ promoter, and K,, for {the ation of Yon its omen promoter. bh. Attime t= 0, production ofX stops after a long peri of prsaction and X con centration decays from is intial steady-state level. What are the dynamics of X snd Y2 What are the response times of X and YP Positive ovdbuck, What is the elec of postive autoregulation on the response time? Useas.a model the following linear equation dXIdt = P+ B,X-aX with P< Explain each term and solve for the response time. When might such a design be biologically useful? What happens when B, > @? Turing off auto-reguation, What i8 the dynamics of a negatively asto-regulation ne once its maximal promoter activity is suddenly reduced feom B, to ‘What isthe response time, and how docs it compare to simple regulation? ‘Two-node positive fedback for decision making. During development (tom an egg 10 an embryo, cells need to make ireversibledeesions to express the genes appropri ate co thei designate tissue types and cepress other genes. One common mechs iso is positive transcriptional feedback between twa genes. There are two {ypes of positive feeack made of two transcription factors. The ist type i of two positive liorsctions X > Y and Y -> X. The second type has two negative interactions X Y and ¥ 1X, What are the stable steady states in each type of feedback? Which type of feedback would be useful in stutions where yenes regulated by both X and Y belong to the same tissoc? Which would be asfil when genes regulated by X belong to liferent issues than the genes regulated by Y2 cuarrer 4 The Feed-Forward Loop Network Motif 4), INTRODUCTION To this chapter, we will continue to discover network motifs iy transcription networks and discuss their function. The main point is that out of the many possible patterns that could appear in the network, only a few are found sigaificuntly and are network motif “The network motifs ave defined information processing functions. The benefit ofthese pain why the same network motifs age rediscovered by evolution again nd again in divers 9 1 find significant patcerns, we will rst celculate the number of appearances of dif ferent pattems in ecal and random networks. We will focus in this chapter on palterns with three nodes (sch as triangles). There are 13 possible three-node patterns (Figure 4). Patterns wit two nodes and patterns with rere than three nodes will be discussed. in the next chapters. We will se that ofthe 13 possible three-node patterns, only one the leed-forwatd loop (FEL) i network motif. ‘To understand the possible functions ofthe feed-forward loop, we need vo understand the vegustion descrited by each of its tor edges. Fach of these edges eat be at activa tion or repression iteration. There are therefore eight possible IVI types. We wil see that ofthe eight possible types of FFs, only io appear in large nusbers in aascription etwork, We will analyze the dynamical functions of these circuits. We will ee thatthe ‘omron types of FFEs can carey out interesting Fanctions such ax the filtering of noisy int signals, pulse generation, and response acceleration, Afiee discussing the common FFL types, we will ask why the other six types of FFLS ‘occur much more rarely. Asking Why will ead us to consider functional dierences i the emmon and rare PEL types. Finally, we will discuss the evolution ofthe FFLs aa2 Cuartins go £ Tevet bey Sande ln ope) 0 >> SN DB >, ob Te De Dp Be [UF 4.1) The erst top athe educ op exapsof srap ih hee nes, (01 The 1 consesed treed directed sgap. The eeorwl lng subgraph 5m esc oop sage 42 THE NUMBER OF APPEARANCES OF A SURGRAPH _IN RANDOM NETWO! _ In the previous chapter we discussed the simplest network motif, self-regulation, «pat tern that had one node. Let us now consider larger patterns of nodes and edge. Such Patterns are also called subgraphs, Two examples of three-node subgraphs are shown in igure 4.13: the three-node feedback lonp and the theee-node feed-forward loop. In total there are 13 possible ways to connect three nodes with directed edges, shown in Figure ‘1b, there are 199 possible directed four-node subgraphs (Figure 5.5}, 9864 five-node subgeaphs, ete ‘To find which of these subgraphs are significant, we nee to compare the subgraphs in "he eeal network fo those in randomized networks, The rest of this section is fr readers interested in mathematical analysis of random networks, Other readers can safely skip to Section 4.3. ‘We begin by calulatng the numberof times that a given subgraph G appears ina ren- «low Brdos-Renyi (ER) network (ER networks were defined i Section 3.2) The subgraph G thot we are interested in hes n nodes and g edges. The fed-forward loop, for example, has n = 3 nodes and g = 3 edges (Fguse 4.1). Other three-node pattcns have between ‘0 and six edges (Figure 4.10). Recall that in the FR random network model edges are placed randomly between N nodes (Section 3.2). Since there are N° possible places to put directed edge (Equation 3.21), the probability of an edge in a piven direction betwee 3 given pai of noes is THE HEED-IORWARD LOG NETWORK MOTIF a 44 p=EN a2 {tis important to note that most biological networks are sparse, which isto say that only a tiny fraction of the possible edges actually occur, Sparse networks ace defined by p the nmer of tines that subgraph G appears in the network, uf potatos fhe des th wgaph tha esa mney ahah tesa non hc nh toe ee pm cl ema he eres the raat an hs the fe fep.2 Ise ties ate perma of he mks el ake sn wpe terete de oe "owe nso |44 mw CHAPTER 4 scales wih the network siz, Image series of lager anu! larger sandlonn ER networks, wectvity A, The dependence af on network size N is ed by scaling relation, This scaling telation describes the way that the number of subgrapts in Equation 4.2.4 depends on the size ofthe network yaoring prefers) all with the same mean cu descr eno = Ne 23) 1 saling relation fell us that tte scaling of subgraph mbes in ER networks depends only on the ference between the mumberef nodes an vdgesin the subgraph, 0 y For example, V shaped patterns, such as patterns 1 and 2 in Figure 4.1b, have nodes and g/~2 edges. Their numer, therefore grows linearly with network size Nyasa NEE 20) we double the number of nodes and edges in the rand network, the number of V= hoped subgraphs wil also double, These patterns ae very cumivon i randons networks, {In contrast, the Fully connected clique (the lst pattern in Figuee 4b) has six edges buat only theee nodes, n= 3. This subgraph scales as NV-K= N* and therefore occurs very rarely in large random networks Let us now consider the case of our two irangle-shaped patterns, the three node feed back loop and leed-forward loop (Figure 4.1). Both have the nodes, = 3, aad thee ‘edges. = 3, and so, using Equation 4.2.4 and the appropriate symmetey factors (a= | for Feed forward anda = 3 for feedback loops), we find Nine MNP aan Njg> = UBB NE 28) This result is remarkable. ‘The scaling of che nutnber ofthese triangles with the network size goes as N= NY. nother words, the uber of these triangle pullrns re constant ‘in ER networks and donot increase with network sie “The reason for the fact that triangle numbers do aol depend on the sie ofthe random ‘erwork i thatthe mumbo af shape pair of edges inthe nctwork scale linearly with ‘network size N (Equation 4.2.6), but the probability that a V-shaped pattern will clase to forma triangle sales as 1/N (beeause an edge that emerges from a node atone arm of the V and closest into a triangle by pointing to the node atthe ether arm needs to choose he ‘one target node out of N possiblities) This yields a total of N/N = N® triangles, This AE 4.1 Mune of Fel ortoté Lop nd eeck Lp ih The Ns inthe Transpo Netwackof ob Uh a aarp thi ok ain Random Ser Fee Tiorvord op (FFL) Feedba lp wih 3 Neds rend | LreLs hes beso moeingrnb ass 745 naane nt si sn usons seh ete nega a ee reans that tangles and more complex patterns occur rarely in random networks. We now turn back to thereat transcription networks 4.3 THE FEFD-FORWARD LOOP 1S A NETWORK MOTI How do the muinbets of patterns in transeriplion networks compare to the numbers expected in random networks? nthe F col transcription network that we use as an example inthis book, there are 42 feedforward loops and ne feedback loops mae ofa cyle of three nodes (Table 4.1. In catras, in the corresponding randomized Et networks with the same mean connectiy- say = S000 = 1.2, the mean naihe of feed-forward loops is only about 2 (Equation 427), Ny Pog ™ DNL and the mean numberof Febuck loops sess than | (Equation 4.2.8), 21-08 Nottm standard deviations of these numbers ace generally the square roots ofthe means, Iv, because in many cases the number of subgraphs fllows a Poston distribution in ER random networks The comparison between real and random networks is shown in “Table 4. ‘We sce thatthe ed-forward loop (EFL) isa strong network motif T oceuss much smoee often than at ranom Hs fequency Is greater than is frequency in the ensemble of randomized netwarks by more than 30 standard deviations. fn contrast the three- ‘node feedback loop isnot a network motif itis actually an anti-moifin many biological eto) Tn Fact, in sensory transcription networks such as those of E.coli and yeast (Le etal, 2002; Milo etal, 2002), a well a higher organisms, the feud forward loop is the only sig- rufcant meework mo ofthe 13 possible thre-node patterns. Im this sense, these networks are much simpler than they could have been. The same conclusions apply also when com- Paring transcription neworks to mote stringent ensembles of randamsized networks that ‘more closely preserve the properties ofthe real network! Tae pr eye te dee see he too: the rf acning ml tig fae ‘rescence ype Ch Rol arg wns ser oh yee46 CHAIR Hct. Tel fared oop nthe ct enact two ck aks partite FL the massive overabundance of feed-forward loops raises the question: Why are they selected against randomizing forces? Do they perform a function that corfers an advan tage tothe arganisn? “To address this question, let us naw analyze the structure and function of the feed-fr war loop network mati 44 THIE STRUCTURE OF THE FEEDFORWARD LOOP GENT CIRCUIT The feed-forward loop is composed of tanseription factor X that regulatesa second tran: scription factor, Y, and both X and Y regulate gene Z (Figure 4.13), Thus, the feed-forward oop has two parallel regulation paths. direct path from X to Z and an inicect path that goes through ¥, The diect path consists ofa single edge, and the indirea path i 2 eas exde of tw edges, ‘Skins See one average noe em a Gl pos Ow le amy Bec in reget ey “nmetl nel, Ty ance tarry ern meer lone a emacs it ors it ma nyse hl meres Nan gs se pre she mero ‘ogame sf hn eo. Dee eta he ee eae he ae ‘noch cn be gsr! mT compe by xaony sci ae fel, een svc ee "ie mayne he tor agua Fars en el nen ty hos ifr dos Ited dpe nr cine nay punta Thine ade ne srt ne ape ‘lon mal orcmpurs bere ete epee perigee ‘en ork 200) ely eer FCs eek TaN} Te Lt 7 if [bi ICU 6 The it sn combinations (ype) off orm eps: Ass Jeo etation sn ‘symbols denae repre ach of the three edges in the FFL can correspond to activation (plus sigh) or repres- sow (minus ign). There are therefore 2"= & posible types of FFLs (Figure 4.3). "The eight FFL types can be classified into two groups: coherent and incoherent. This ‘grouping based on comparing the sign of the direct path from X to Z to the sign of the indirect path that goes through ¥. In coherent FFs, the indirect path has the same ‘overall sign asthe direct path. The overall sgn ofa path i given by the multiplication of the sign ofeach arrow on the path (so that two minus signs give an overall i sign) For ‘cxample, in typest coherent FFLs, X activates Z, and also activates an activator of 2, 50 tha both paths ae positive, {mn incoherent FFL, the sign ofthe indirect path is opposite to that ofthe direct path For example, in the type-l incoherent BFL, the dtect path i positive and the indirect path is negative. The two paths have antagonistic effect, Note that incoherent FFLs have an odd mumber of minus edges (one or thee). Not all the FFL types appear with equal frequency in transcription networks (Figure 44), The most abundant FFL isthe type-l coherent FF. (CI-FEL), in which al theee lations ate postive (Mangan and Alon, 2003). The C1-FFI, will be atdied in detail i this chapter, The second most abundant typeof FPL scros biological networks is the incoher- ent type-t FFL, (I-PFL) (Ma et al, 2004; Mangan etal, 2006), which we wll aleo study in etal. ‘the sx other FFL types seem to appear much less frequently than the CI-PEL and the N-EFL, Toward the end ofthe chapter, we will ry to understand why the frequencies ofthe FEL types are eo different. In addition to the signs on the edges, to understand the dynamics ofthe FFL we must ‘so know how the inputs from the wo regulators X and ¥ are integrated at the promoter Df gene Z. That is, we need to know the input function of gene Z. We will consider two biologically easonable logic Functions: AND logic, in which both X and ¥ activities need "9 behigh in order tw turn on Z expression, and OR login which either X or ¥ issuUO GOO GUI 1. Relative sbundenee of he eight HEL types the ramscption network yt ad eh 8, ype are meet Cand for cate and sncaezet The ent bed the Egy egal databases and has aboot twice 5 ny edge asin the network Rgue 2) (Pn Magne cient. ‘thus, there ate eight types of FFL sgn combinations each of which can appese with a east two types of input Fanetions (AND, OR}, After noting the signs and input functions, we need to consider the input signals 0 this ireuit. The transcription factors X and Y in the FEL usually sespond to external stim ‘These input stimuli use represented by Uhe input signals §, and S,(Figare 4.5) kn some systems the signals are molecules that directly bind the transcription factors, nd in other systems the signals ate modifications of the transcription factor caused by signal trans duction pathways activated by the external stimuli, The effect af the signals which casey information front the exte at world, usually operates om a mach Faster timescale than the wansceipional interactions tn che HF When S, appears, transcription factor X rap idly becomes active, X°, binds t specific DNA sites inthe promoters of genes Y and a manner of seconds, and changes the transcription rate so that the concentration ofthe protein 2 changes on the timescale of minstes to hours. ‘We will next discuss the dynamics ofthe proteins that make up the FEL a funtion of time following a change in an external signal. We will begin with the most common FFU type in which all unre interactions are postive (Figure 4.5). As forthe input func tion of the % promoter, we will fist consider AND loge. "This isthe case in which both activators X and Y need to bind the promoter of Zi order to initiate the production of protein Z GUT 4. The ey HL waa AND tng urn Yo a Yo Seong fontvnses tte let KandY tthe po FUR 4 activated by sip Sy hich ease 48 DYNAMICS DE TIF COHERENT TYPE-T FFL WIT AND LOGIC Sppose thatthe cll eepeeses unterous copies of pratein X the top transcription factor inthe FFL. The input wX isthe sigaal 8, (Pigore 4.6). Without dhe signal, X isin sts ina tive form, Nom at tims = 0,4 eng goa Sieger te activation oF X. "This s kBOW asa step like stimulation of X. Ass eau, the transcription factor X rapidly transits to Atsactve form X* The active protein X" binds the promoter of gene Ys initiating produ tion of protein ¥, the second transcription factor in the FFL In parallel, X* also binds the Promoter of ene Z. However, since the input funetion atthe 2 promote is AND logic, X" alone cannot activate Z production 1nd Y*, hig means thatthe concent Production of Z regutes binding of both X ‘lon of ¥ must build to sulicient sels to cross the activation threshold for gene 7 ‘Tis activation thresh is denoted K, In aldion, Z ctivaton, reales that the second Y* (Figure 46). Thus, once the Inpa signal, i present, 20 dat Yi i its active forsignal S, appears Y newds accumulate inorder to activate This resuts in delay in 2 production, ‘We will now mathematically describe the FFL dynamin oder to see how 3 snp ‘mathematical model can be used to gain an jotuitve understanding ofthe function of ‘ene circuit, To describe the Flt ususe logic input functions, Peduction of Yaccursag Fate sen X* exces the activation theesldK,, a described bythe step function Drodiction rate of ¥ = B, 8(X* > Ky) sy ‘When the signal S, appears, X rapidly shifts to its ative conformation XI the signal 48 strong enough, X* exceeds the activation threshold Ky nd rapidly inds the ¥ po niote to activate transcription, Thus, ¥ production begins shor ily aie §. Tae acCuola: tion of ¥ is described by our now familiar dynamic equation with tera for production anal another term for degradation/dittion: A¥AdE = 0.00 > K,.)~a, ¥ 432) ‘The promoter of Z in our example is governed by an AND gate input function, Thus, ‘the production of can be described by a product of two step functions each indicating "whether the appropriate regulator crossed the activation threshold Production of 2,0 (X*> K,)O(Y*> Ky) 459 ‘Thus, the C1-PFL gene circuit has thee activation thresholds (aumibes on the arrows in Figure 4.6) Im the case of strong step-like stimulation, X* rapidly crosses the wo thresholds! K,, and K,,. The delay in the production of Zis due tothe time it takes ¥" to ‘sccunnuate and cross its threshold K,. Only after Y* crosses the thesholl ‘on proceed at rate 8, The dynamics of Z are governed by a degradatica/dih And! production term with an AND input function: Hike = BOK > KOO» Ka, 2 aan ‘We now have the equations needed to analyze the dynamics ofthe CL-FEL. We next ‘analyze its dynamics as sigu-sonsitve delay element 4.6 _THVE C1-HFL 1S A SIGN-SENSITIVE DELAY ELEMENT ‘To describe the dynamics of the C1-FFL, we will consider the response t steps ofS, in ‘whieh the signal is irs absent and then saturating S, suddenly appears (ON steps), We ill also consider OFF steps. in which S,isat first present and is then suddenly removed. For simplicity, we will assume throughout that the signal S, is present, o thatthe tran scription factor ¥ isin it clive form isis ol Ka wn 2 a he fcton ofthe frais iss vere acct cosas bad ayy an abe ne pnd 3 Toe yey 6) ON Step of §, 0 Yt ye produced at rate i. Hence, as we saw in Following an ON step of Sy. ¥* begins tobe pro 7 sme ‘Chapter 2, the concentration of ¥ begins to exponentially converge tots steady-state level igure 4.7 4611 olay Following yey Ger) 462) Recall thatthe steady-state concentration of Y is equal to the ratio of is production and degradation/dilation rates: Bye, 463) 2 a AND input foci, n which one ht seu Pod of governed by an AND ve lp i ea on 5 ie ep a gh in AND pt These pu Yokes me scum a 0 Stan od Tht, een oni ae by tie) The dlp etme ned fe" fo ech thes ad an be se trshaly sth ine wen he onsration coset hol net Mh Ky The ey, ean be nd sng gun 462 % Yop) Yall eo) =, asa‘his equation can be solved foF Ty yelling Toy = Hay fog MO = KJ] 459 “This equation describes how the duration of the delay deperas on the bidchemical para eters of the protein ¥ (Figure 48a). These parameter ae the lifetime af the protein, 3, al the sitio between Yy and theaetivaton threshold Khe delay can therefare be tuned evolutionary timescales by mutations that change these biochemical parameters, [Note that the delay Toy diverges when the activation threshold K, exceeds the eal state level of, because pratcn ¥ can never reach is threshold ta aetivate Z. Recall that Ys prone to cell-cell fluctuations de to variations in protein production rates, Hence, robust design wil have a threshold Ky that i significantly lower than Yyy 0 avoid these Nhuctuations. In bacteria, K, Is typically a least 3 to 10 tines lower than’ Yan! typical parameters give delays" tt range fram a fee minutes to few hours 462 No Deby Following a OH Step of ‘We just saw that Z shows a delay fllowing ON steps of S,, We now consider OFF steps of Sy In which §, is suddenly removed (Figure 4.8). Following an OFF step, X rapidly becomes inactive and unbinds from the promoters of genes ¥ and Z. Recall that Z.is gow cerned by an AND gate that requires binding of both X* and Ye thesefore only takes one Input to go off forthe AND gate to stop 7 expression, Therefor, after aa OFF step of Sp Z production stopsat once. There is no delay in Zdysunaes afer an OFF ste. AG.b Tho CHEF fea Sign Sensitive Delay Hemet ‘We saw that the C1-EFL with AND logic shows a delay fllowsing ON steps ofS, des ‘ot show a delay following OFF steps This typeof behavior scaled sig-sensitive delay. where sign-sensitive means that the delay depends on the sign ofthe step, ON or OFF. ‘A siga-sensitive delay element car also be considered asa kind of asyinmetec fier For example, consider a pulse ofS, that appears only brielly (an ON pulse) (Figure 4.8). ‘An ON pulse that isshorter than the delay time, Ty, lags not leat any 7 expression in the CLF. that is because ¥ docs not have tine to aeetimnslte andl eros ts activation treshoid during the pulse. Only persistent pulses (lange that Vy) Feslt in Z expe son, Thus, ths ype of FFI is a persistence detector for ON poles Cn the ater and i ‘responds immediately to OFF pulses. In contrat to the FF, simple regulation (with no FF.) does not iter out short inpat pulses, but eather shows producti Jong a the input plee is present ‘of Z that lasts a6 od Si ‘Why might sign-sensiive delay be useful? For clues, we can turn to the uses of sign-sensitive delays in engincering. In engineet= ing sign-senstive delay is commonly use in situations where the cost of un errr i not ‘symmetric, familiar example occurs in elevators: consider the bear of light used (0 sense obstructions in the elevator deot. Ifyou obstruct the light with your hand, the dloor opens. ITyou remove your hand for only a short pase, noting happens (that isa intive Delay Can Proteet agains Brie Input Flaetuations a "Hu 4 eyo ein ie CEL te te odie he {esergion ar Thee Tn made desl ylang wh the degradation te EY es ft of heat of he station eo ys he mal ey 8) level of, dmeed (4 Dyauiies of fe CLF. faoning an OFF sep Sate = 0. Al prod: tion sd grain Fates ae sua 1) THe caren tpt Ps eh AND lpi so penne dete. Ait ple al 8 oes a ge ehmugh Une to scimitar eatin rosa for 2 Hance, Zp tv exprlae A Perse pe eZ ration a del poco ope with no delay whan, veo, a Shon Der l, 2002) titted out). Only ifyou remove your hand fer a persistent length short pulse of light oftime do the doors close (@ persistent pulse of light lends toa respons) Put your hand back in and the dou's open immediately. Again, the cost of an error (loors closing or ‘pening atthe wrong tins) is asymnvetric the design sims e respond quickly toa personBaw CHAMIER GLE one) Jnthe beam and make sure that the person has moved away fora persistent period of ime before closing the doors. ‘Ihe sign-sensitive delay serves a protective function, In transcription networks, evolutionary selection may have placed the C1 FPL in diverse systems in the cell that require such a protection function, Indeed, the evwironment of calls soften highly fluctuating, and sometimes stimuli can be present for bi pulses that should not elicit a response. The CL-PFL can ofera filtering Fonction thats advantageous lo these types of fluctuating environments. The conditions forthe natural selection ofthe FEL based on is filtering funetion are discussed in more deal in Chapter 10 4.6.5 Sign Sensitive Delay i the Arabinose System af Fol ‘Our discussion ofthe function ofthe PEL has dealt with this gene cite in isolation. In reality this network motif is always embedded within a network of addtional interac: ‘ons. It is therefore crucial to perform experiments on the FFL within living cells, to see ‘whether it actually performs the expected dynamic! factions. Experiments have demonstrated that sigh-sensitive delays ate curried out by the FF. in ving cells. For example, dynam Behavior of at FT. vas experimentally studi in wellcharacterized gene system in E.coli the system that allows the ells to grow on the sugar atabinose, The arabinose system consists of proteins that transport the sugar arabinose into the cell and break it down for use as an energy and carbon source, Arab ose is only used by the cells when the sugar glucose is not presen, because glucose is superior energy source and is used in preference 1o most other sugars. Thus, the arabinose system needs to make a decision based on two inputs: the sugars arabinose and glucose. the proteins in this system are only made whea the following condition is met by the sug: ars in the environment of the cll arabinose AND NOT glucose The absence of glucose is symbolized with the cel by the production cf small mol cecule called cAMP. To make its decision, the arabinose system has two transcription activators, one called CRP that senses cAMP, and th athe called ara that senses ara on ae gh Ei or ow i” ne oad o PACU 4 apse dai th CPL te ate fi heat ‘sem encodes crays ia ize the spar aabinsse orAD) nn ransor ate the el aPC ab thespian X= CHP lg Scala eee! inthe ‘2 upon glaoue aration) snd inthe pence of = arabia by the stvtor Y= ANC The Ip funciona AND gate Az cont este wis 0 (empl ult the experien se the lc peron,a which same slater X= CRP repulse the latte operon tt X dee notre Y= Lc ‘Dashed rows Raph on ranriptonal elk Togs in which te onput ane pdt act he i ‘al eg by tansperting the wpa tothe ell and degrading) () Dynamic af the prom ity Sftheara and le aperane were ronored thigh tere reeton in rowing el My mee EEN Sorescet protein (expend fom he eva promote ote presence of Sy Te experien ‘owed he dynamics ater ON i OFF stp oy, Sn GFP per all org aie aia eel. A ly ecu FFs ser ON tps bt at aor OU sep Calon Mangan et 20) ose. These regulators are connected in a C1-FFL with an AND input funetion (Figore 493) Theinpat signals inthis system are S,= cAMP and § Experiments on this system used steps of S, and monitored the dynamics of the pro ‘ote of the arabinose degradation genes that act as node Zin the FFL. delay was found sfier ON steps of ,- but not afier OFF steps (Figure 4.9. The delay in this FF, following ON steps of, is Tay ~ 20 min under the conditions ofthe experimen! = arabinose. ‘The observed delay in the arabinose FFI. is on the same order of magnitude as the Aeration of spurious pulses of the input signal S, sn the environment of F. col. These SPurious pulses of , oceur when F. coli transits between different growth conditions.56 w CAPT us, the FF in this systerm may have earned the typical timescale of short octustions lonthe input + them oat. I responds only to persistent stimuli eh ae persistent periods of ylacose starvation that eequre wilization af the sugar arabinose goal and ean [Note thatthe Fk i the azabinose system shows sign-senstive delay despite the fat {hat it embedded 9 ston sul a pratein-levelfeelack loops (hig 9), although the thewry we have discussed concems a shave gene FEL circuit in isolation, the arabinose FFL shows the expected dynamics also wher embedded within the interaction networks ofthe eel, Abs Te OR Gale CLI Is a Sig Seaitive Delay for OFF Steps of ‘What happens if the CL-FE, has nt OR gate atthe Z promoter instead of an AND ype? ‘With an OR gate, 2 isactivatedinumediately upon an ON step ofS, Deeaus i only takes ‘one ipput to activate an OR gate. "Thus, there is no delay following an ON step ofS, ta contrast, Z is deactivated a delay folowing an OFF step, because both inputs need 10> fff or the OR gate to be inactivated: ¥* can activate Z even without X* and ic takes tine for ¥* to decay away after an OFF step of Sy. "Thus, the CL PEL with an OR gates also a sign sensitive delay clement, but with sighs eppesit to those ofthe AND version (exercise 42). shows a delay after OFF steps, whereas the AND version shows a delay after ON sep. Hence, the OR gate CL-FEL can maintain expression of even ifthe input sgl momentarily lost Such dynansies were demonstrated experimentally inthe Nagella system oF E cal using high-resolution expression messurements (Figure 40). This FFL contrals the production of proteins that sel assemble into 8 motor that rotates the flagella that slow Ecol to sw, We will discuss this system in more detail in Chapter 5. The delay observed in shi FEL alter removal of, is about one call generation time — about | under the eon ‘ions ofthe expetinent. This delay is un the same order of magnitude as the time takes ‘oassemble a fagella motor The OR gate FEL, provides continued expression for about an hour ater the inp signal goes off and can thus protect this gene aystem against ian sient Inss oF input signal 46,7 Item Summary We have seca that of the 13 possible three-wode patterns, aly one isa significant net “work motif in sensory transcription networks that need (a rexpond to external stimuli Ihis network motif s the fed-forward loop. The FFL has eight possible types, cach co responding 10 spoctic combination of positive and negative regulations. Two ofthe FFI types are fae move common than others in transcription networks, The most comaot form, called coherent type-t PPL, sa sign sensitive delay clement that can protect aguas, unwanted responses to fuetuating inputs, The magnitide of the delay inthe FFL. ean be ‘Ri nti inst tcl am ones saunas ps the rat sre pei el Oh okies AD) dete sate toe ise Thorsen gv athe prance tr HURL €10 the CL-FAL with OR gn the pla aye of Ecol. Th output ges ch iL [NDPOR, nck wp he gaat The np aul areca tors a lene in a> ‘ion ere pete an tempers that fe! he pomater fhe aetivaor HRD. hep al 5, tothe een ects, i, chek oil that sere who heist or ae completed pron Inka of Fa cll lp ic exported hough he ates cu of th cl) (8) Experi o the ro ‘ter act of heap yn uted by mea fae fusreseprokinexpeed seep fete it yronotn ara, ON sep ff) Proc Synami ar an OFF tgp’ the presence sf, The esa ae st othe wildtype foes acer which the gve Ft FA was desert geome The PL. gensateradly aaron OF ep’, (Hom Kale! a, 2008) tuned over evolutionary timescales by varying the biochemical parameters of regulator Protein Y, such ais bifctime, maximal level, arl activation threshol, 42 HE INCOLFRENT TYPE-1 PHL - a Wie will neve ta fin thy client FEL te atin the faction ofthe incoburent fed for ard loop network motif. We will see thats can function 38a pulse generator and sign- Seashive accelerator, 471 The Structureof the Incoherent Leto analyze the send most common FH. type, the incoheret type PEL (H-FFL) The LF, mout mates wp about third af the FFLS in the transcription networks of ‘2b sd yest igure 4). “he IL-EFL is made of two parallel bot antagonistic regulation pubs, tn the W-FEL, SetvatorX ativan 7, bat i alao actives Y — a eoprestor of 2 (gure 4.08). Thus, the "wo ams othe H-8FL act in opposition: the direct arm activates Z, andthe indict ainSaw CuAPTER f on or Lt sememetin LO wie ” GUE.) @)Theincoberes type FEL with an AND gate athe Z promate, The inputs aresigl nd, The epson thesbld of gne Zy ep fy (0) The bd sate gle {or the promoter egon of 2, regulate by acter X and reese Transeo ere whe the ‘stor Xs Bod, and ta mach leer extent whe both dtvator and rprestor¥" ne band. The AND Input fenton thar corepondsioX" AND NOT represses Z. The gene Z shows high expression when the activator X*is bound, and much weaker expression when the repressor Y* binds (Figure 4.10). To analyze the dynamics ofthis motif, we will continue {0 use logic input Factions Hence, the dynamics will be composed of transitions between exponential approaches to steudy states and exponential decays. As we saw above, these piecewise exponential slynamics make graphical analysis and analytical solutions eather eos 47.2. Dynamics of the 1-FFL: A Pulse Generator ‘The 1-FFL responds tothe input signals, and S, (Figure 4.113) Upon a sep of, protein X becomes activated, binds the promoter of gene Z, initiating transcription and causing protein Z.to begin tobe produced (Figure 4123). tn parallel, X activates the production of Y. Therefore, after a delay, enough protein Y accumlates to repress Z production and Z- levels decrease Thus, the II-FF. can generate a pulee of Z production, Let us analyze this in more detail, Consider the response toa step addition ofthe signal Sim the presence ofthe second signal S, When the signal S, appears, protein X rapidly transits to is active conformation, X* ‘Ihe active transcription factor X* binds its DNA site nthe ¥ promoter within seconds, and ¥ begins to be produced. Since S, is present o FICUNE 412 ( Puedtihe dams of he HPL lowing an ON sepa 0 the presence of 5, The lap tp occurs att = am ap toto activo Asa esl Ze be expres Imada ie reprensor protein ¥ pred, nd cretally repress production when eromes the ‘epeson heel eg tn fatal plato a dey rater are equ (PA of een ‘renin the pulse ke amis ofthe FEL Shows the ypamiset Ziman incoherent pet FL ‘th repeson cele = 35 an 20,The repre oe the ratio fhe sendy tt expres Sho nthe seen of epesr to the sealystate expen ws aie Fes Ty the He when ees ep, the protein Y is in its active form Y* and accumulates over time according te the produe- ton and degradation equition60m CHAPTER 4 Vt = 8, =a, ¥* ar Hence, ¥ shows the familiar exponential convergence 10 it scady state Vy 4s y= ¥G-e%9 an suldiion o activating ¥, molecules of X* also bind the Z promoter As. result, peo tein Z* bens to be produced st a vapid eate since its promoter i occupied by the et ‘ator X* but there is ot yet enough eepwessor tthe cel to inbibit production (Figure 4.120). ln this phase, a7 B02 (a3) and Z accumulates, beginning an exponential convergence wa high evel 2 = Ba: 1) =Zq ney while YK 474) “This fast produetion of Z lasts until the repressor Y* crosses its repression threshold for Z, K,. At this time, the production rate of Z {in our logie approximation) suddenly «drops to low vl Tn the extreme case of no leakines,t drops to = 0. The onset ‘of repression occars atthe moment that Y* reaches K,,. This pression tie, Tyy ean be found from Fquation 4.72 by finding the time when Y"() = Ky. showing that, depends ‘nthe biochemical parameters of protein Y Yay log (U0 = KN ars) At timesaler‘T,, the Z promoter is bound by the repressor Y* an the produetion rate of Zs reduced. Figure 4.12a shows how Z.conceatation decays exportemtilly to.3 new ewer steady-state Z = Bla, (ce solved exercise 2.) 2A) = gt Py Zyheore Ved x0) where Zs the evel reached at time'T,. given by Equation 4.74 at. etn (a7) ‘and Z, isthe final steady-state 2 level, due the low expression lovel when both X* and Y" bind the 2 promoter: ara) The shapsof the dynamics genes sd by the 1-FF1. depend on, thebasal production rate of Z. This basal rate corresponds to the low rave of transcription from the repessed promoter. The eee of different basal levels.on the dyanuics is shown in Figure 412b for egos Ba » FIGURE 4.15 Response of the TPL shorter hail regulation that ech sae te say fhtfevel Simple replat, dase nes CEP, ful in, (0) Reyponc ime af the -FFL as fanction of ‘herpes caeicent = he ete of warereied to repressed 7 exes. Alo sown is the 9 Pld ssp tie ofp replatia yy = bg. several values ofthe repression factor F; defined as the ratio of the maxianal and basal activity ofthe Z promoter, alo equal tothe ratio ofthe unsepressed and repressed steady- state levels of ro)Ow GHAFIEK + ‘When the repressor as a steong inhibitory effect on 7. production, thats, when F >> 1, 2 dynamies show a pulse-ike shape, In the pulse, Z levels fist increase and then decline ‘wallow lev. The I-TFL can therefore act asa pulse generator (Mangan and Alon, 2003; Basu eta, 2004). 12.5 Me HFT Spesds the Response Tine In addition to pulse generation, the {I-FFL bas another property it ean accelerate the response time ofthe system. You can see in Figuee 4.13 thatthe response time of the I [FFL fs shorter than that of simple-regolation circuit tht raches the same steady-state level of 2. Uhe response time can be found graphically by the tine at which the dynam ‘ho use a laa ie allay 10 dhe steady-state Level (dase lines is Fijane 4.120). [Note that one cannot speed the response time of the simple-regulation circuit by increas: ing its production rate, cause such an increase would lead 10 the stendy slate level. The II-FFT. achieves its fst response time by initially using 9 high production rate, and then using the repressor Y to lower the production rate ala delay to reach the desired steady-state level, ‘Toanalyze this speed-up quantitatively. let us calculate the response thme T. the time te reach half ofthe steady-state level. In the 1-FFL, half steady state is reached during the initial fast stage of Z production, before ¥erusses its repression iareshold. Thos, the response time, Tyas found by using Equation 4.74 by asking when the concentration of levels reaches halfvayt0 7, ‘unwanted increase of wa Lal azo) hich can be solved to give an expression that depends on the repression coefficient F Pui Tha Ha, log (2H amy [Note that, as shown in Figure 413b, the lager the repression coeliient Fhe faster the response time becomes (Ty, ~ (2a, Fa large F). tn ather words, the stronger the effect ‘of Yin repressing production of Z, the faster the performance ofthe [L-FFL compared to a equivalent simple-regulaion circuit X-> Z made to reach the samesteady-state level of 2."The response time becomes very smal! when F >> 1, approaching Tyz™ 0. At the oppo- site extreme, the limit of no repression, F= 1, we find Tue log(ia, * Axmcnioned previnsly. he eespons tae enna he smaller tha the minimal ie tak proteins bet Senoedand tate hia bw mina aan ten ag mame ee Table 2 arte tent ate cant eae are iy epootion op ‘eriouaten hight thee a premcel eerste which is the same as the response time for simple regulation that we derived in Chapter 2. ndeed, when F =I, the [-FF. degenerates int sinple-regulation cirult because the repressor Y has no effect on Z, and the edge between ¥ and Zs ponfunctional 4.74 Response Acrelecation fs Sign Sensitive In contrast to the accelerated responce seen aller ON steps, the response after the signal S, is removed accurs with the saose dynamics as fra simply regulated gene (no acceleration ‘or delay), In both simple and 1-FFL cireits, OFF steps ofS, lea to an imsmedite shut. down in Z, production, Ths immediate esponse to OFF steps inthe II-FFL is due to the AND logic of the : promoter, in which unbinding of X*issulficient to stop production (igure 4.11H). Aer production sts, the concentration of protein Z decays exponen: tially according to ie deprndation/dlation rate, Hence, no specdup i found in the OFF direction relative to simple regulation, “Thus, the T-PFL is a sigrsenstive response accelerator. Sign-senstive means that response acceleration occurs only for steps ofS, In one direction (ON) and not the other (OFF) I-FFLs with OR gates have generally the same function as those with AND-gates, bt accslerste OFF and not ON eesponses. 4.25 Fxperimens Stuly of che Dynamics of a 11 FFL ‘An experimental study of response dynamics of an II-FPL is shown in Figure 4.4. This experiment employed a well- characterized system, which allows E.coli to prow on the sugar gulctose as carbon and enesgy source. As in athor sugar systems, the genes in the ‘alacose system are not highly expressed in the presence of glucose «superior energy source, The galactose wilamion genes are expressed at alow but significant level when both glucose is absent and galactose i absent, to allow the cell o grow rapidly on galae- tose should it appear in the environment. When galactose appears, the genes are fully ‘expressed, The galactose genes are regulated by an II-EH, with the activator CRP and the repressor GalS, High resolution measurements show thatthe response ofthe output genes is accelerated upon glucose starvation {an ON step of S) compared to simply rege Inted genes (Figare 4.14) Removal af the repressor interaction in the II-FI, abolishes this seceletation, Im aaddition to studying this network motif within a natural context, one can study It by ‘making a synthetic I-FFL made of well characterized regulators, Weiss and colleagues constructed an I-FFL using the activator xR as X, the repressor CI of phage lambda as ‘Y.and green fluorescent protein asthe avtput gene Z (Basu et al, 2004). This “synthetic ‘ircuit” in B, coli showed puse-tike responses to steps of the input signal S, (he inducer ‘of Lusk), The synthetic construction of gene circuits isa promising approsch for isolating, and studying their properties! "pth ge cele ate review i (Misty ab 20: Spr al lowe, 2005) tapes ince stn (Garde ts 3090 Bele 20 Rrra, 208 You 2940 lator sah te rere Sit ow ter 2409 tn i Tb) and asa ane Nn, 2 Yaa 1202 fant 2 esa ea, 2808)marin ett genta) NL Byam te 8 ne ane st Ten es a seein ths ot a ich ire ca Th odin a pon hic nls Dams te pl pm wea) an fy en pac GY een eh pote Nr GFP ae ferafer OM wep var tee are) 8 the absence of palactone, Also shown seme Nich the GalS site in the guil:T® pronnonce was delet hernia {ne 7 gc) Thao fy mid pane nen here the ‘ ie ofthe al on aie gt gun Coe Magn tal 206 476 “the Wa Spt geno en Sn fe have by now seen three nen ee by rc ferent ways apes the sponse time of transrpion seis The bern ha ie pose tine of aero gion co sou he noe natin ine rp al re aes me ne toes ply eternal sal thet wap sponse times of yene regulation that we have discussed are: “ 1 mcrae gradation rte a Chai 2, th eons tne salon nr porta te etn eee s(2Va, where ais a sum of the rate of specific degradation of the protein and th fle of dton by gots = ay, ay terete, renin the epee tn else epics hae sae san fhe este X= Bsa os eee eum me obs fects of incrvased degradation a. This creates a futile cycle, where th ‘an ipl proued and ep deed ths lca beset een soe sens dete the cnt of ised paactat doe tthe Boe of speoding the esos tine‘ incre spt apis oth to ur ON and en OFF of gene expression. 1. Neato autoregultion: As we savin Chapter 3. negative autneaton eam speed cis by a lage feo. ‘his speed-up is dec the ability to uses strOag PO- renter large peat cat 8) 10 give rapid inkl production, and then to 10 rocton of by slepesson lic the desed sted tates eased Noe thot Fry rn ON is spesded trn- OFF snot bot father has th sate OF? response eas simple regulation, The negative autoregulation strategy works any Fr peo vm tat eat repress themselves, nantly, ly Rr asririon factor proteins, “L Ineaierent EFL‘ Incolverent FF. can signilcanly peed p ON responses, a8 pasta inthe previous section. this i de (nial espe preduction that i ter Teena iby o dlyed ecpresor, to achieve a desired stenly state. ‘this speed-op Spies tothe low auction state inthe presen of, Itcan be used 10 spect the teaponse ine of any target protein, net only transcription fasion Designs 2 and 3 can work togetber with Is lage degradation ete can farther speed the response of negative autoregulation and incoherent FEL 4.8__WHY ARE SOME FFL TYPES RARE, __ ‘We have o far examined the structure and fonction ofthe two most common FE. types ‘ie will now ask why the other six FFL types are rae in transcription networks (Figure Ua) To addees thin, need ta consider the steadystae computations performed by the FELs We wil see that some ofthe race forms have a functional disadvantage in computations: they lack responsiveness 10 one oftheir two inp 4.8. Steady-State Logic ofthe H-ATL: S, Cam Tarn an High Expression ‘The FP. has two inp signals, S, and $, Upto now, we have considered changes only i done ofthe two inpats ofthe FEL, namely, Sy an staged the dynamis in dhe presence ‘tte second init igual, What happens inthe IL-FEL fe remove 2 The signal Sy nuscs the repre ¥ to ase fs active form, Y*, and ind the promoter of gene to inhibit te eapresson, Wher, isremoved, ¥ bconses inactive and unbjds from the pra rnoterof gene 7, As aresll, Zs nol repressed and is expressed strongly (Fagoe 415 “The realting stoauysuate egie nf the ILEFL with an AND gate line in Tale 4.2 “The second inp § basa strong efecto the steady-state Fevel of Zs matting iy 2 factor F= By, 48.2. tf, a Ranly Selected Circul, Nas Reduced Functionality ‘as mentioned above, not all FEL types are found in equal amounts in transcription net Noske Artng the incoherent FFLs far example, the mt common form i 1-EEL(@bout So ca0ss of known Fla) wherces the other forms are rare ([3-FFLs and M-TFLs are total ess than 585 of known FES), Why? To adden this question, we wil focus on two very similar structures {1-FET-and M> PEL, th sieuira hoe two activation arrows and one repression arcow (Figure 4.16). Theoo = CHAPIIR | tive ava ropromo nd the tinction pan 7, shows a merase Wa hgh warepressed seal sate (ans ton) ‘only difference is that in the I-FFL, X activates Y, which represses Z, whereas in the Me FX represses Y, which activates Z. "The minasand plus edges in th path have simply changed poston. How ean this subtte change result in sucha large ei ference in the appearance of the two circuits in transcription networks? ‘he structural dfferonce between these two circuits means that in the I-FFL, X activates both ofits target genes, whereasin §-FFL, X activates one target, Z, and represses the other, Y.Can a transcription factor be bath an activator and a repressor? As mentioned! in Section 2:3 the answer is yes: transcription factors such a the bacterial glucose sarvation sensor CCA agtivate many target genes, but st ab repressors for other yenes. The molecular mech: anism i often simple to understand (Collado-Vides etal, 1991; Ptashne and Gann, 2002) ‘in bacteria, for example, an activator often binds a site that is close to the binding site of NA polymerase (RNAP), helping RNA to ind or to start transcription once it binds. the activator binding site is moved so that it overlaps the space occupied by KNAp, binding ofthe activator protein interferes with binding of RNAp, and the activator act as repres sox. Similar features, where transcription factor can activate some targets and repress fhers, are commonly found in eukaryote regulators, though tve dete mechanisms an vary thus, K-I¥L sea biologically feasible pattern. ‘What about dynamic behavior? Is H-PFL a slgn-sensitive accelerator and pulse genera tor as wel? The answer, again, eyes. Is easy to see that upon an ON step of Sy Z-bewins tobe produced vigorously, activated by both Y and X. At the same time, since X represses rect regulation EN A. Steady tt Hepuns ofthe 1. Vk Combinations fit Sg . 23 "1G 1 Te inborn p64 AL ad ype FPL, Y, the levels of ¥ begin t0 drop. When ¥ goes below its activation threshold for Z, the production rate of Z decresses and Z levels decline. This yells a pulse-ike shape of the dynamics (Figure 417), just s in the -FFL, with a speed-up ofthe response time. The: ‘magnitude ofthe speed-up relative o simple regulation is the same as in HPL, When ', ges OFF, Z production stops at once (veto the AND gate, just as in the case of I= FPL. Thus, H-FFL isa sign-sensitive acelertor It hasall ofthe dynamical capabilities of ILFRL in response to, signals. The same applies to [2-FFL and I3-FFI. (except that they accelerate responses to OFF step). What, thea, might explain the difference inthe occar- rence of [I-FFL.t and 1¢-FFLs in transcription neiworks? “The main difference between the two FEI. forms isin their steady.sate logic We sa shove that L-EPL responds to both 5, and S, tn contrast, the steady-state oulput of H-EFL does not depend on ST se this, note that when S is present, production of Y is repressed and its concentration declines. At steady-state, protein ¥ isnot present a functional levels. ‘Therefore, when S, is present, §, cannot affect Z production, because Y — the detector pro- tein for $, — isnot present. When S s absent, on the other hand, is OFF regardless of ue to the AND logic. Thus, § does not affect the steadystate level of Z in H-PEL. The M> FFL isnot responsive to one ofits two inputs (Table 4.3) ‘The lack of esponsiveness to one ofthe two inputs may be one of the reasons why I= FFL s selected less often than 1-FI The same reasoning applies also to 13-FFI. Similar conclusions apply to the Fare and common forms of coherent FFs. Coherent type 3and 4 PFLs have the sue sign-senstive delay properties as the much more common TAQIE 3 Steady Snte Ost inthe -FFLand 1 FL ra amen the lp Sigman, 3 PAS Zia FP a ° ° ° ° 7 ° 7 ° : ° ° a ' ° 1 0 1 ae. Bye, 0 tow.B'6, u » 1 eh BA i i 2 um B's, 0 tow 8'sa, ' 1 0 tow. Be, ‘Nein iqg aac a ticerse WAT ce ll Fos pl in oe 2 Ta oa rk Hab ie nar alts, WoevaxeanbGUE 7 Dyaamis ofthe H-FFL flowing a atep af Stn he pctence a, poten Xs active wn stvates predictions repre the roetiono¥ Wh ¥ eas sles bao he el oct Iie the mtenteaton of boys dra Proucton a dey res ane y= 2yea,= eand t= 1a heal pest hugh Iypecl coherent FFL, However these types with AND logic cannot respond (othe input signal S, forthe same reasons discussed above Note that we have analyzed here only AND gate inp factions, and pot OR gate, “The discussion for OR yates is more complicated because they can have multiple intr ‘mediate sates of Z. FPL types 13 and 14 with OR gate can in principe be vesponsive to botis inputs, Similarly, the [2-FEL with AND logie is just as responsive as the (7-FEL It fs an interesting question why these circuits are not commonly found in transription networks, 44) _ CONVERGENT EVOLUTION OF How does evolutionary seliction act on the thrve regulation edges Inthe FHP Its Fea. sonable thatthe most important function of the regulators X and Y isto respond to the signals S, and S, and accordingly regulate Z, "thas, che lirstordee selection i for the simple Vishaped structure wlhste X and ¥ regulate Z (Figuee 4.180). te the third ‘edge, the edge from X t0 ¥, that needs special explanation. Recall that it only takes one ‘a few mutations in the binding site of X in the ¥ promoter to abolish the edge X > Y, it does not add useful function (or iit actualy destroys Fonction) this edge will ‘apidly be lost in evolution In the cominon FFL types, CLPEL and 1)-FFL, we have scen that the edge between X and ¥ can add a fenction, such as persistence detection ar ple generation and response acceleration, Presumably, such fanctions are usefl enough in some systems to select his edge: Mutant organisms without the edge ace lost fram the population de to their Ucereasel fitness, On the cher hasd, ia 4FPL, adding the edge between X and ¥ can suse ss of fanctoslty ~ the enlist longer sponds 0S,‘ mig ‘ihe saxhan edge tobe dug evaton, ee ow ld Fs we The msconfig ocean cot bere to gees wi sr feo se Fo a an neo ge Sanna ed ins uncom dere of ssuence sie bese te gees. Sc ese do be hoogos, * ‘Did FFLs evolve in a similar way, where an ancestor FFI. duplicated) and sconce pes 7s ett hans 9 ot et or ep nol und Z ino organs ae oe bth replated by FFL in espns a ra mrt aol he wo FFs ha commas aectr FF, he epson Tuy neo Fa wl soe nog Howe he reo ey 7 oxntpann sh FF pis gre 41 Te eqns of eeu ston aaa at iy ery blog t complete diferent trnsxtion fate ies aaa tn sesitobave comer independent on tesa oon ce a Pony eaes (Conant an! Wage, 2003 Bab el, 2008, Presumably, the FFL isrelscv ase en baci performs an poste fanein i the dle rganims se pmut one ea vlan nd selection of Ls lb dscsedin Cage 10 410 SUMMARY. ee ‘We have seca that sensory tranecription networks havea measure of simplicity. OF the 13 possible thvee-gene repulation patterns, only one, the FFL, i network motif, Purthes- tore, of eghn posible FL types, only two are commonly found 2° FAGUNT Ain seen ofthe. th Val pen in which Xan eget ee Saas elon bp Ths om Xt Gon cane ees taal a adiiont itive delay, acceleration, puise generation. (b) la cee tilomt dma! scion tg. up sestie ty, cen in aaa ee Zant en rinse bread aL ego he MSU tlc ann fea crm teagan aaa tee naa hn th agen ofan car FFL Zn ere ee est: Fina chscnee nts, Ho eat i see eee w fete he pvesiae acannon ace ehe two common PP types ean carry out specific dynamics! functions. the coher. ent type-t PEL (CL-FEL) can act asa sign-scnstive delay clement, "This, ican function 852 persistence detector, filtering away brief fluctuations inthe input signal. With AND logic, brief ON pulses of the input signal ae filtered out, whereas with OF lic, brief OFF pulses are filtered out This function can help protect gene expression in envirensnents with fuctoating stir “The second common FH. type, the incoherent type-1 FEL (I-FPL),can act aka pulse gen eatoranda response aceeerator. This acceleration can be used in conjunction with the other ‘mechanisms of aceleration, sch as increase degradation and negative autoregulation. Some types of FFL have reduce functionality clative to other types. Inpatticula, some ‘ofthe PF. types cannot respond fo ane oftheir two inputs This reduced Fanetionality may ‘plain a least yar dy why these PEL ypes ae relatively rare in teanserption networks ‘This chapter didnot exhaust al of the posible dynamical functions ofthe FFLs, ‘hese Kbit sepa by a eC tea pte seat once oa Tm ohn ume of ate eget Zeesion 448. The diamond agun. The diam pattern occurs when X regulates Y and 7%, and oth ¥ and Z regulate gene W. Analyze tbe 10 types of diamond structores (where cach edge is ether activation + or tepresion -) stitheespect to thelr steady-state esponaes tothe inats 8,5, and Sy Use ax AND faput funetion at the W pro- pater, Do any diamond types lack responsiveness to any input? To all three input? 449. Represilator Three repressors ate hooked up ina eye 'Y Zand ZX. What ee the resulting dynamics? Use initial conditions in while Xis high and ¥ = Z Solve graphically using logic inpot functions. This circuit was constracted in Docteria using three well studied epessors, one of whieh was also made to repress the gene fr groen fluorescent prtein(Howita and Liber, 2000). What woud the Tesulting bacteria look like under a microscope that dynamically records green fluorescence? 4440, Interconnected PPTs, Consider a coherent type-t FFL with nodes X.Y, ane Z5 which, inlined to anther coherent type-l EPLin which Y activates Y,, Which activates 2, fa. Sketch the dynamics of Z expression in response to steps of the signals SS and Sy (Steps in which one of the signals goes ON or OFF in the presence of the other eignale), Can the dynamics of the interconnected circuit be understood ‘hated on the qualitative behavior ofeach FF. in isolation? 1. Repeat forthe ease where ¥eepreses Zs that the X, ¥ Z-FFL isan incoherent {pet FEL. Assume that Y binding to the Z promoter can alleviate the reps: Ingettect 0°charter 0 Temporal Programs and the Global Structure of Transcription Networks 5.__ INTRODUCTION oe ‘We have seen that transcription nelworks contain recurring network motifs that can perfoemn specific dynamical functions. We examined two of these motifs in detail, ato regulation and the feed-forward loop. tn this chapter we will complete or survey of motifs in sensory transcriptional networks. We will se that sensory transcription net works ae largely made of just four families of network motif: the two we have studied, including fed-forwacd loops with multiple outputs, and two families of larger motifs ‘The larger motif families ace called single-input module (SIM) and dense overlapping, regulons (DORs), “The SIM network motif fsa simple pattern in which one regulator controls » group of genes. Despite its simple structure, the SIM has an interesting dynamical function: it can generate temporal programs of expression, in which genes are turned on ane by one in a defined order. In Fscherichia cali, these temporal programs are found to match the functional order of the gene products This isa "just when-needed” production strategy, not making a protein before it s needed. Such a steatepy is optinnal for rapid production ‘ofa system mde of different types of proteins, under constraints of limited resources for producing these proteins. More detailed temporal programe can be generated by a generalized network motif, related tothe feed-forward loops (FFL) we have examined in the previous chapter. his generalized moti is an FL with multiple output genes, We will se that the multi-outpat FFL can generate temporal programs with diferent orders of activation and inactivation of the genes,HG The sgsipt ae (SM uo mein fac X veges goa {Gat fone no alin tpn acts. X nly san ete welt A exam S96 ie spline bry ating (nthe ai spa uns ee ees) ke last mot family, called DORs (or densely ovstlapping regula), i dense ‘of regulators that combinatorially control output genes. The DORs can carry out de sion-making calculations, based on the inpat functions of eacl gene Finally, we wil discuss how the network motifs fit together to bulk the global struc luge ofthe transcription network. These four autf families appeae to account for vets ally all of the interactions in sensory transcription networks. 5.2 _THESINGLEAN! ‘The network mot 1 MODULE (SIM) NETWORK MOTIF we have studied so fr all had a defined umber of nodes (one node in the autoregulation motif three nodes in FFL), We will now took for larger motifs, Fach ofthe longer motifs correspon to a fanily of patterns that share a common architec theme! Ihe first such motif family found in txanseription networks is called the nat module (Figure 5.1), oF SIM le srt (Shen-Orr etal, 2002). ty tater nenscripion factor X contre a group of target et Zp Zap Zy (igure 5.1). ach of the target genes in the SIM has only one input ho other transcription factor regulates any of the genes. In addition, the cegulaion signs (cctvation/repression) are the same for all genes in the SIM, The last Feature of the SIM is {hat the master transcription factor X is usually autoregulatory. nro yn counting oltre hap inetng om: if pose sabi A eh sgt cts hand (ree nang ot wh ne Ce ol ble soe Ang motes pnt “he StMs ave a family of stractuses with afc parameter, he numberof target gees ru They area sirang network moti when compared to ranvrs networks! hiss because fpisre to fia in randee networks node regulating, say 1 otcr nodes with no other ‘edge going into any ofthese nodes. Despite thei snap stature, we wal sce ha SUMS tum out to have suteresting dynamics What isthe fsa of tM? The most portant tak of SIM is to contr geoup af genes according to the signal sense by the master regulator Ihe ges & SIM ays faves common hitlogkcl Function, [or example, SIMs often eegulte gens that participate ina specific metabolic yuhway (Figure 5.2). These genes work sequentially to asenble a ina kin of molecu: ested molecule atom by ato assembly line ‘Other SIMs contol groups of genes that respond (0 2 specific stress (ONA damage, heat shock, ete). These ones produce proteins that repair the different firms of damage caused by the stress, Such ates response systems usally have subgroups of genes that specialize in certain aspects of the response. Fil, SIMs can control groups of geDes that together make up a protein machine (sch asa ribosome), The gene products assen- ‘ie into a functional complex made of many subunits 5.3._ SIMS CAN GENERATE TEMPORAL EXPRESSION PROGRAMS Tn addition to conteoling 2 yene module in 2 coordinated fashion, the SIM has 2 more subtle dynamical function, The SIM can generate temporal programs of expression, in urhich genes ae activated ae by one ia defined ones, [A simple mechanism fr this temporal order is based on diferent ehresholds of X for cach of the target genes Z, (Figure 5.3). "The threshold of each promoter depends on the specific bining sites of ia the promoter. These sites can he slighty different in sequence and position, resulting in dilleret activation thresholds for exch gene, When X activity ‘Tang sep encom Rants Reena mee ai Ch levt godt fn hae dyer etic tation se oie) ht Fan st er ‘Scexpnety vues oa hn many rect he an connec hay 1 asa Bae yt Inconel tence og dt ey she Tcl symone esa appends The we cen eon Exuoran ue itgese ach il lars oc fo smc ta eon coe timgeccre cde Sagres he alter se peta eto {DPR Choshin pees! icioa mths Tove uprieuawerersee ate o DEAN, ht ae Raisins onan a PRA Seen pacar spe for deen mencaat ttt amanctteroneter ha een acne axa conarion o ‘dennis carne to Wag ere ‘Nett eh ol past pty ns SI chesney he mae later ge Sa Fortune yen hg plac vt esha ah pth pts fr by oes fenced anno no ihe mae gee ne nthe ee tach srteaes tcl nd een hts ya he rac the eoebe ene ‘hae tne nee peste more he gos rere. ie eptine eibace op Oe a wenase idueea ohn sfrgnne heh rt smc mech 6m ar thn th ther Sie deancapton sean etens alee nar rane) Tsoi eae oop ao reece eee ee ucamesn senting ned hope 67a mw CHAPIER 5 aX _F. 2 8 + ED 5 <5 ES as) GUI 2 A singleapa mule SIMD vaulting there setae pay The mai ssn ser X ress a ton af geen henner ey yard Each on iret peri). Thee yes etre th eoesion af sare 10S 19S alsin nha, 8 The spe X- 1 bd to X an reste the prota that in tine es Xn ic ids the roms ope the potion of cazynes Ta ches mai ecb a mbt high lel eal is rediton in ete podtion changes gradually in time, i crosses these thresholds, K, a different tines, andthe genes are turned ON or OFF in a specified onler (Figure $3) ‘When the activity of X inceeases gradually, i rst activates the gene withthe lowest ‘hreshold. Then it activates the gene with the next lowest threshold et. (gure 5.3). ow= ever, when X activity goes down, the genes are alleced in reverse order. Hence, the first. gene activated isthe last one to be deactivated (Figure 5.3) This type of program is alled ‘8 lastinfirst-out(LIEO) order. “The faster the changes in the activity of X, che more rapidly it crosses the different thresholds, and the smaller the delay between the genes. In many syiems, there is an asymmetry in the naturally occurring dynamics of the regulator ativty. For example, ‘sometimes turn-ON is fast, but turn-OFF is gradual, In such eases, temparal order will be more pronounced inthe slow phase ofthe transcription factor activity profile? Experimentally, temporal order is found In a wide variety of systems in E. cli with SIM architecture, This includes metabolic pathways (Zaslaver ct al, 2908) such as the arginine system) (Figure 8.4 (See color inset fllowing page 112) and damage repair sys lems such a8 the SOS DNA repair system (Ronen et al, 2002). The genesin these systems ate expressed in defined onder, with delays on the onder of 0. generation hetween genes {about 5 1 10 sn). Tene nance coder ean tat are mic fer ha the rte tine fhe new we he ge ge pao Fre sii gee Xo nce by ameter eth tate orca aint agian 8 ‘culng iin he concent of pcs X wi tA vcd srmnon rape cer Iisqoened ys edbech areal of he co te downed enon ges Fore he sunt attes Keane the abl pe of tab ay eure ie ‘hc metal poic cn chang slot sens a na eeee Invaninywttecarenaniuoey dw iene in heene rf mendes nao an yey nthe ele aan Inte tec f eter uptime ae e Sirs bing extra spe ihe es mary mies wl he bay eng a gm jot he an wi on coer Hc 5:3 the Sit can generate enpal pogins expression, Ashe ati OF pay ricki ‘ces the erent tess fr esc ag! poner na ded oer The gone with he lowe ee ‘1 is tuned OM Fie the gene mse ho rr? turned ON tant Wh X a hy dies. eos the theta ern oe ais ob), 4s thece any meaning to the temporal order found in the SIMs? In E. colt metabolic pathways, such a8 arginine biosynthesis, the following principle unifies the experimental findings: the earlier the protein functions in the pathway, the earlier ts gene i activated, ‘Thos, the temporal order of the genes matches thei functional order. This is an eco- omical design, because proteins are not produced before they are needed, Such 2 just ‘when-needed production strategy can be shown, using simplified mathematical models, to be optimat for rapidly reaching a desire Nux ofa metabolic procht under constraints f prodvcing « minimal total number of protein enzymes (Klip etal, 2002; Zaslaver et a, 2004)" ‘The precise temporal order generated hy a SIM can be varied by mutations tht change the relative order of the thresholds ofthe genes, Far example, motations in the binding Site of X inthe promoter ofa gene ean change the affinity of tothe site, changing, the theshold (Kalic and Alon, 2004) This suggests that the observed temporal order is main tained agninst mutations due to the selective advantage afforded by justshen needed Production strategies, Temporal orders found also in damage repair systems controlled by SIMs, In damage "epair systems, turn-ON is usually fest, hecause the regulator needs tobe activated rap- ‘idly in ord to rapidly mobilize all repair processes. As damage is repaired, the inpt sig nal ofthe regulator declines and the genes get turned off gradually, reaching 50% oftheir ‘maximal promoter activity at diferent tives. inthe systems that have been studied, the ‘genes responsibe forthe mildest orm of repair are turned of first, and thote responsible for more severe damage repair ae turned of ster (Rosen eta, 2002}, Tn ay pens ead prec the eer he prin oats hep the phrase ahs ph 8, Paine 200,a + Temporal order also characterizes a lage numberof other global cellular responses Fxamples ince genes tieed throughout the cell cycle in bacteria (Laub etal, 2000; ‘MeAclams and Shapiro, 2003) and yeast (Spllminn etal, 1998), genes tgolated by dif ferent phases ofthe cicadian clack that keeps trick of the time of day (Young, 2000% util et al 2002), a8 ell as genes in developmental processes (Dural aad Powryuie, 2002: Kmita and Dabok, 2003). Ta these global well-tinned Ponses, genes are often regulated by a minster reyulator lated by additional regulators responsible for smaller subsystems, ‘Te Doral order may he genecated by the ation of a master cvoidinating regulator even ifthe network patter is nt strictly a SIM, in the sense that it has more than ane regulator, The Present analysis of temporal order applies sls to circuits with multiple regulators, a ong, call regolstors except one have a constant activity ducing the interval of intevest and also How did SIMs evolve? There are may examples of SIMS that regulate the same gene systems in different oxganisms. However, the master regulator in the SIM is often diler centimeach organism, despite the fact tha the target gooes a highly homologous (this tal, 2005, Vinay etal, 2005) this means that rater shat dpication of an aneesteal TEMPORAL PROGRAMS AND HL GLOWAL STRUCTURE wy SIM togetter with the the same relation pat stor to create the modern SIMs, evaktion has conve 2 i the diffrent organs, ust a8 we saw tht evuliion comerge on the FFL network motif (ee Sect 43}, restnably the SIM regulatony pat tera is rediscovered anal preserved inst mttions because sis uefa) eo selected In short the SIN: can generate jstwhen-nocded temporal prograsns. AS was men tioned above, the SIM circuit generates LIFO order the activation order of the goes fs everse with respect fo the dewetivation order (Figure $3, However, fm many cases, it Seems more desirable have an ation adr that isthe sa the FIFO ender is desirable for assembly processes sthedeactivaton anc (FIFO) order) at requte pas ina defined order, some Promoter ttened on i also the list torn olf irs in-fiest early and some late in is ease when the process is de-setivated, ii etter forthe early ienes to be turned QFE before the late yenes. this FIFO order prevents waste feomn ned lesly producing early genes proteins alr late ones ave OFF, We next describe cirewtry that can achiove FIHO axder. To describe this circuit, we first discuss generalizations of network motifs and an adeltionat important matt family sn sensory transeripion net ‘works the mu-output feedforward In. $4 TOPOLOGICAL GENIRALI/ATIONS OF NETWORK MOTIES We have so far discr ss relatively simple network motif, When considering larger and "ore complex subgraphs, ane is fced with large numberof possible patterns. For exan ple, there are 199 possible four-node dinected patieens (Fire 55) and over 9000 five node patterns Thereare millions of distinetseveu-nade patterns, tn ore to ty 0 gtoup ‘hese patterns into families thot share a funetional theme, one ean define topological ‘generalizations of motifs (Kashian ct a, 2004), To describe topological motif generalirations, conser the faniia feed fosward loop (PPL) The FFL isa thtee-node pattern with noes X,Y, and Z (Figure 5.6. the simplest form of topoloyica! generalization is utained by choosing 2 nods, say X, and duplicating 5.60) The resulting pattern #8 a double-inpot PT Tis ean be repeated tn obtain mle snput FFL. goseralizations, There are two other smn itatoog with all of ts edges (ig ple topological genetliations of the FUL ind by splicing UH called mult-¥ and srut-output FFs (Figuce 8.6 and 0) nia es, {In principe since the FFL is network mati, any of these the patterns could also be network motifs thats, any of them could eccur significantly more ten than in randoun- ned networks. However, only ane ofthese genenaeations is actualy a motif transcrip: (FEL tion networks The chosen generalization isthe ‘We can slow ak why. What might be the function of the multi output FFL? To address ‘his question, we will consider & wel thot it can generate a FIED temporal program, in contrast t the LIPO ner generated by SIMs havactsind case of the malt-output FFL and seeOPPO OHS | am ‘a pom lvaaion | cea SEP OKHX OOD ' Leone e noe - GEOL MYOE&OD ‘ PRA GESEOSO . roth Dany ana ve ,eoere Bos POR OSS YO “etchooy LESCOL SHR OOS YUASA OH DOS CODD DO BYE EMRE HRO DS CEES OPRSS Cte bee wo se Vid hae A Doe DO 1 apleainton othe skate FFcand ao tes The FO) Geral be Selig mo Mohn tlanens on en 8) CONDADO OE SERCO BE 5.5_THE MULT-QUTPUT FFL CAN GENERATE FIFO TEMPORAL, ORDER CLUE HO OLY thotconte ie podeton olga Eee cond mrs When Bc in rae enironent wih abundant net, dds happily and doe EVES OPENS terme When conan bene wn clues dean pow sv en ‘meter-size motors attached to helical agella (propellers), which allow it to swim, Its also. EEL YYGobesd feverstes a mvigation sytem hat st where to gon serch fo beter ie Copter 7 lla dt south naigation hemota)system. We wl now conse he GENE O oa OH fevestnt maketh parts gl nerwa CHIAIIER & ne BOR LIE Meee os i ae tae a Niwen “Balen Ginna CUI 4 me Magee mae ol ands senses fo Mash 2008) “The tagella motor is a $0-no device built of about 30 types of protein (Figure 5.75, see also Figure 73) The motor is electrical, converting the energy of protons moving in through the motor to drive rotation’ at about 100 Hz. The motor rotates the aellim, ‘whichis long helical filament, about 1 times longer than the cell tis attached to (E cal isabout 1 esicrom ang) Flagells rotation pushes the eel forward a speeds that ean exceed 30 micronsiec. “the motor is pat ogeter in stages (Figure 5:7) This san amaring example of biologi ‘al sclFassembly, like throwing Lego blocks inthe sir and having them asenble in 3 house. ‘the motor and flagellum have a hollow centeal tube through which the proteins rave to assemble each stage. Thus, each stage ofthe motor act asa transport device for ‘he proteins inthe next sage. ‘We will focus on the tansription network that controls she production ofthe motor proteins The proteins that build up the flagella motor are encoded by genes arranged in ‘six operons (an operon is group of genes transcribed on the same pce of miRNA). The flagella motor operons are regulated by two transcription factors, both activators, X and Y¥-'the master regulator X activates Y, and both jointly activate each ofthe six operons, Zi. Py ory This regulatory palterD isa malti-output PEL (Figure 58), mtn pup tnt the enpenure monmote ace® HNGURI 5 Sebonati an fhe muon HF tt eles he gs mtr gn Show bic opt anton cach ptr th ptton eno X= INDC, Wl i In this muli-outpt FFL cach uperon can be activated by Xin the abience of Yan by Y inthe abssnce ofX Ths, the input functions aes to OR gates! Eaperiments sing high-esolution gene expression measurements fond tat he st fagell operons shows defined temporal order of expression (Kali el, 201 Kal and ‘Aon, 2000 (gure 59 (See coor insert allowing page 12). When the acer seas he proper conditions. they activate production of protein X the concentration oF X galt ally increases, and asa esl, the Z genes gt turned ON one by oe, with bout cel {generations between them. The order in which the operons ae tured oni boat the Same asthe order in which the proteins they encode participate in the asely af the motor: st rng in he iner membrane, then aod, a secoad rng. Tithe pin ple of just-vben-neded production that we discussed inthe single-input modal (51M) network motif the temporal order matches the functional order af the gene products “The SIM architecture, however has a limitation, as mentioned bef: the sn- OFF coe is evr its respect othe tora-OF ode (as infos, o LO onde) {re 3 In contest the SIM, the aye turr-OFF onder is the sae the tre-ON Crd: he Gi proc urna on lao the Bet turned off when Dal ae Tonge needed. In other wos, the genes show airs in-rs-ea (FEO) order How can FIFO cider be generated by the mult-output FFI? The mechani Is easy Co understand (ure 5.10). Recall that inthe Fogel spt, X and eect function in OR gatelbgc atthe Z promoters. Thus, X alone i Scent a tr the genes ‘Wi we se ga pe ry he inp! aye ean ane SUM pt fen) frat sen 2608) ce ecg he conan he rer dt ‘fi pose i te pe h eacs rcote ym arn tr esp ma tach m pose araion sume pert ne ey Cal dey eu i eo ny users eal pl kee bse nt se by eels Stn magento saiusoceat saanLGA 9 (eer nse allowing age 12) Tena bars are the onic expe iw and els gh pcm Ae Tt aa ieee ih eon ee Sei mtr a sey mo ost nausea teas He ee as scbratclyoo ei Te Yona teens psc watnce fine ee ee con. therefore, the turn-ON order is determined by the times when X crosses the active ‘on thresholds for the promoters of 2,, Zt. These thresholds ace K, Promoter with the lowest K i turned om fist a last Figure 5.10). 1 this were al, Kops Ky The the one with the highest Kis turned on nes would be turned olf in the reverse order once X Tevels decline, eesulting in LIFO order, just like in the SIM. But here Y cores o the res «ue, When production of X stops so that protein X duction only scays aay Y esl around spo : stops when X decays below the threshold fr activation ofthe ¥ promete. Ka, Thereafter the eves of protein ¥ gradually decay by degradation Since ¥ is still present afer X levels have decayed, the turn-OFF onder (in a properly designed FF) {s governed by ¥, which has its own thresholds foreach ofthe gene KsKy'y in KA TIFO otde is achieved if the onder ofthe thresholds ofY is revered emmared to that of X. Thats ithe X thresholds ae uch that Ky «Ke 2 the ¥ thresholds ate arranged so that," »K. 2 (igure 5.10, Thisis the design that i experimes and » $0 that promater 1 is curned on before 0 that promoter 1 istarned off before ly found inthe Naga system (Kalle Alon, 208). the temporal onder in this system was shown to changs by mutations ho alfccted these action thes, 5.51 Te Mul Outpt EF Can Ao Ae 6 Fenton Dect fr bach Output snaition gemrating a FIFO temporal or, te mop FL als conveys alo thefonction tefl fora Top that we dcssed in Chapter 41 pots each ste capt nodes nets em th sign sete Ser property ofthe FL or ra Sica th gly, the I fonctns ava device tht dys he econ oe along te or of X eins deere in ection 466) ther word tna bor OF ple of allowing esc on Wed Xe one fora pester eogth oie “atone Fon hrtoe provide an nine ipa. allow res sen ent oi fi meted inher th i Grin gases found tebe on te rr acl eration wih tote ine nce compte the esemly of Rela ter Kali tl, 208 ‘asta speaking ach procction faction could be afl lage produc renee theremin el sik toencuner seating esronment hic can eeu podeton art cat ovr ne, eso ml envionment tr ‘Sola ie roots of he ee that encodes X The OR FL cam ens tht ugly eereton spr oly when the appropriate conditions have been sensed peste Teegrnige A raaene dentition of the X promoter wouldnt be sce tum val roto Foe mloat FL. the peer of th FL that ccs mot en ean actors We will th et hap tha ther Nae yeah fren 7 geeatratons Te mn Leon races cca gral dani he hate did88m CHAPTERS snd or ney me nt 50 Te Chap aio se cpe ican gente FLO tga ee of expres Topang herr oscatn oie rte we eget ean 56 SIGNAL INTEGRATION AND 6 FRATION AND COMBINATOR HSIANS AND DUNSE OUIREATPING RIGMLONS ‘We now complete net ens 7 Bow cm oy finer iin ary arin cnr ce ewok ins strep, Pts ath ses multiple outputs, and SIMs. Our fourth cn ay dS han final network moti family stems eons the Weare montaned tha ths are 199 pose tigers ey een svt pin ee eatnkng simply bene hey cota ca etn of spore ps of oa th iat Po sotwork tate np nth tata ct ping elton pater ered th fn ie 10. the bf, two Input tansri we spt transeription fectors, X, and Xz, jointly regulate two ourput genes ‘The fan ges re citing ed eat Jens ban etn ct dene oetonageslnn selontt iss elle dene oedpying regulon sone Wy a given transcription factor) s Se a pon to BOR or shor igure 8) Shes the DIOR is you af inp transcription factors that julate 2 set of output gener in Ip oeaping sey DR kha sy meh ing nh an i ae ‘ond thiol dimes! fenton at nepal tte oma Sh tenet neon 238 The DOR can bein favs ee ad PS g EE Pi semana enh cers sphcldaiuies indchoerotetaneagenasatyopccioae ‘he okt ctl ys a so eho cb or ee ages comes es Sh, 208 DI QAO ia SEK ec ntegr pees wi ot ans no ts ‘AGURE 512 TRE Dion ene snaking devi 1 uncins as an aray of ges Gap fantons) ha NERS multiple aps to compute the regulation af each output ene re spi netwone such 26 hose af ctl and ye several large DOR: carting en ta undceds of ens The gees in ach DOS Ware shared global coach sess response niente oF osetess ‘oF key clases of ‘ereem ponents Oe, lel rgulatr governs many a (NEY sopplemented fg ormerousseglstoos tha regulate bss of hs genes YE ‘example, in cli the lal regulator CRP senses glucose starvation 28 OES! wth multiple transcription a ean caseni diferent sagt determines which separ ties genes ae at Fr i path sat te einen The DORs for the ‘backbone of the Tretwor’ global scuctare, as we will se nes 5:7 NETWORK MOTIFS AND THE GLOBAL STRUCTURE OF SENSORY TRANSCRIPTION NETWORKS. _ ‘Webive desribed thefour main ne Jrotffamilies in sensory transcription tworks redeyeltio, fed aeward Toop, STs, and DORS ow are est iotifs positioned ma eectusenh oer nthe network How much ofthe neon oe they cower? te ne quentions, one eco ww a mage af the nebo: 1 dlisficut 10 dena anomlen nse nan understandable way Figure 2.2) Hoven network moti se apt produce a sgl ess completed image Posed 99 the following coarse- ining prover To draw the network, replace each “occurrence ofa SIM that regulates sien by square marked with he uber, RePace Se rmultioutpat FEL by a tH neared withthe number of ouput genes Repos ech DOS ‘with a rectangular box Tecupeits inputs tpt and connections The resus intricate plete, but Ca nett the lool network ste igs (Gee color fnsert following page 12? “The eours grained networ ‘susahowrs that senvory transcription networks such 2 ia layer of DOR, The DORS form a singe lyer—they sc obtained by this procedure, such a5 that shown i gute hose of E coléand yeast are made ‘do ot fore cascades, ‘That is criti ch Seana a Teh nn en oan 8 a Crm ar yan gi eo nl core cn can eel YG Fr tt tc eve ae ire ses Sheet 208 ——_—_—EGUI 511 the dose ovpping eons (DORs) network net and an eam te el ates respon and saioary phase (aumbeg 198) CPt Shen Oe 2005 there is no DOR at the output of th wother DOR. Ths, most of the computation done by twork is done a a cortex of promoters within the DOR. the hyer of DOR also contains most of the other motifs the FFLS and SIMs ate int. brated within the DORs, Many of the FFLs are multi-output, with the same X and ¥ rep lating several output genes. Negative autoregulation is often integrated within FPLs and also decorates the master regulators of SIMs, Overall the rather simple way in which the network motifsare integrated makes it possible to understand the éynamies ofeach motif separately, even when tis embedded within larg patterns Virtually all of the genes are covered by these four network motifs in the oxgaaisms studied sofa, including the known part of the sensory networks of bactris, yeas, worms, fruit les and hurmans (Harbison et al, 2004; Odom etal, 2004, nm et ab, 2004; Bover tal, 2005) Thus, these network motls represent the major types of pales that oecar in sensory transription aetworks A striking feature of the global organization of sensory transcription networks is the telative absence of long cascades of transcription interactions, X-» Y -> Z-», ete, Mast Benes are regulated jst one step away from thee transcription factor inpurs Why are long cascades relatively rare? One posible reason is response time constraints ‘We have seen in Chapter 2 that information transmission down transclption easeades i slow: protein ¥ needs to accumulate fo cross the threshold for regulation of gene 2. This accumulation time is on the onder ofthe lifetime of protein Y, usually on the order of ‘many minutes to hours. Thus, long cascades would typically be far too sw for sensory ‘ranscription networks that need to respond quickly to environmental stresses and nutri ents, Sensory transcription networks are “rate limited,” with components that are often arvana oct 04 i fl Same Ion tom She GUT 5.4 (Se aloe insert flowing page 12) The ghia scr opt of he tor ps pre rnp rt ea th sip oe ee Patgeiesand operons Rectang are DOR. Trans ar opt single re oupet of SIM Bc and ved lines corpo to active sd eepresion in Orerst aaa) ited networks slower than the timescales on which the network needs to respond. Rate the network ar active ude whieh stir Soc ste im yas sues YO Ty Ts summary, seoryirensrpon networks ars anne open 10 WS aw satel “ sdes ace found in stent eal ore Io‘seades are far more common than long cascades. Must ether patterns, such as thees nxt feedback loops, are conspicuously absent! Hence, the ubgraph content of these net ‘orks is much simpler than it could have bees. They sees to be bull of w sal set of clementary eitcut patterns, the nctwork motifs. FURTHER READING Shea-Orr 8, Milo,;R, Mangan, S. and Alon, U. (2002). Network motifs i the transcriptional regulation nelwovk of Facherichia cl Na. Genet, 3: 64-68 lenput Module and emp Order Jab MIT, Medan 112, FeldlyumT, Fraser. CM. and Shape, L 2000), Global analysis ‘of he penetc network contollings hacen eal ele, Sconce 390-268-248, MoRdamns,Fth ond Shupito, L. (2003), A bacterial cell-cycle rpultory network opraing in ‘space and time Science, 301 TN74-7 ashes An May, AE, Rosenberg, KR, Dashkin,P, Sere H. Tslyuk, M, Surette, MG. ane ‘Alon, (2008), Jus intime tansription gengean neta patimay. Nat. Gene. 38 496-49, Generalizations of Network Motifs Bey J: and Lasig M. 2008) Local graph liguotent and motif search in bislogeal networks Prot Nai ead. Si, US.Ay 10: 14689-94 Koshan, Nu Mekowits S, Milo, Rand Alon, V (2004). Topological generalizations of network ‘mot Phys ev, B, 70031909. ‘Multi Oulput FELs ane Temporal Oxler Kali, and Alo, U. (2008). Using » quantitative blueprint to reprogram the dynamics ofthe Magela gen network. Cll 117: 719-720 Cascaue Steueture of nseription Networks ‘Hooshangi,S, ThibergeS, and Weis, R (2008. Ulra-censvity aad aise propagtin ina sy ‘ete tanscriptioal easeade. Pro. Natl. Acad U.S.A, 102 3586 Pedraza, 2M, and van Ovdenaardea, A. (2005) Noise propagation in gene networks. Science, 307 1965-9 osenfekN. and Alon, U (2008, Response delays ai the steucute of tansclpion networks ‘Mol. Blo, 329: 645-654 "Ree fick bps ae fen unable como property of patie ops mia (mn ‘un perf psa thunderace pes nanos dane toons eee se espns (Pa, 298) Sey terpenes ayo hee soe {ope tDALin However hy hncoven ewer raga ner tnceeree ae Shree Simp DT meat aon = pale tonne fe norard ep 4 mates aa | BAND oe Blearetiat em i “sponse aceeratin eo, IS ola a ae Se mts, my deen Duwcovnupring SRL itp monacha volectosa’® | [xd FUR 525. Newock mi in seaey tansetion newark. panosts — Si Fu ning Coie’ SM conta By epltrX that cts dwt fer Zz eh wth K tte 0 Xp ob ped Sts ee Deh thes sch hat Tego ae treo on ae ether teu eras se gin fanetn.98m CHARI 52. oust siming 4. For the system of exercite 5. ste there biological reasons that favor placing the Uhesholds K, much smaller than the maximal level of X7 Consider the case whch x iam activator that begins to be produced al time t= O,and consider the ceflects of cell-cell variations inthe production rate ofX. Would a design in which X isa epressor whose preuction stps at time t= 0 provide more robust temporal peogeams? Explain, Solution 8. Imagine that K, i close to the maximal level ofX, X, = fa Since the production rate of X varies from cell co cel, some ccs have higher than. others over their entice generation time (Gee Appendix D). ence, these ate cell-cell varia tions in Xy- In cells in which production is low, we might have X, «Ky: in these cells X* does not cross Ky, Therefore, the downstream gene Z, is not expressed, and the cells at a disadvantage, Thus, designs that provide the required tim {ng and in which Ky is much smaller thar the meun X, (salle than the lowest expected X, given the variability in B) have an advantage let ws conser the case in which K, are much lower than X- In this cise, X «crosses these thresholds a early times and we can ase the approximation of i- ‘car production X()~ Bt (Equation 24.7). Ths, the activation tines ofthe genes, found by asking when X() = K, are t= K/B. ow thresholds thus ensure that all genes ean be activated despite the noise in production rate. One can tune K, and (to achieve the required timing, Note that can vary from cll to cell fB varies, factor 2 change in 2 change int, The relative order ofthe tre ON events of the same cell would, however, not chunge, ‘the activation ti would lead t a fact different genes in a SIM 1. When X production stops. it decays X() = (Bia) ©*. The tuen-DN time of gene His the time when the level of repressor X goes below its threshold: Xi.) that 6 12a Mog A 0 ogee [Note that appears only inthe logarithm in this expression, so thatthe activa ‘on time tis quite robust with respect to isctuations in production rate, more se than for an activator whose concentration increases with tne (where, us we sawabowe, (,~ KB). “This increased robstness might be one reason that repressor ciscades are often used in developmental transcription networks (Chapter 6) (Rappaport et als 2008), 53. 54 55, 56. TEMPORAL PROGRAMS AND THE GLOBAL SIRUCTUKE a 95 I mal-oudpat OF FPL In a muli-cuput CEL wth OR ge logie at the 2, Per acaecatserinton fctorX begins obe produced sa constant ve Brg ie Tee production rate 8 suddenly becomes equal to tere Caleving he downstream genes’, What ate the delsys between pene! (oe What re the topologiclgneraiatins ofthe dimond aa ation fhe damond pattern -» Yp XY Nee BY» 2) based on ipicaonouingealeandaledecaee hae these diffrent from DORs? What are the topolog ‘ . Or Yr Fy» edo Mee Seana, et Yori Yo? Mono tse fvenodepnertanin cee ifs ithe nent caplet SIM with ego: wht the efit of the icf asoration on he master an ‘ton fcr Xin aS Pt chal and ca ea te aT gs ina gen St wih poste mena eee ‘on ef, and no autoregulation ofX. Explain when each desig might be sector Bin dynam Coss ian in hich aves nn we statis nd utes fn Top ga o%, Sy appears tine eoend watts age Cea natal Sm appre sand ace ‘cs of the promoter activity of 7, and by, ne eaNDinlORiogaremeccch sven that the input Funetions of Z, and 7,oe etarren 8 Network Motifs in Developmental, Signal Transduction, and Neuronal Networks 64 _INTRODUCTION . {nthe previws chapters we considered network motifs in sensory iranseeption networks. ‘We sav that these retworks ace rade of sal eof network tif each with define function, We will now ask whether network matis appear alo in othe kinds of bolt cal networks. “The sensory transcription netsorks we have studied are designed to rapidly respond © changes in the environment, I this chapter, we wil ist xara scription network, ane that governs the fits of ells as an gg develops into a multise- lutar organism, of more generally as a cell differentiates intoa different ype of el. This type of network i aed a developmental transcription network, “The main diffrence between sensory and developmental transcription networks is the timescale on which they need to operate and the reversibility oftheir ation. Sensory transcription networks ace wo nike rapid and eeversible decisions on timescales that are usually shorter than cell generation time, In contrast, developmental nctwork oft ced to-make irreversible decisions onthe slow tnueseaie of one or more cell generations ea diferent type of tran- ‘We wil sce that these diflerences lead to new network motifs that appeat in develope tal, but notin sensory, networks. In alton to tanscription networks, the cll uses several other networks of interas- tions, such a6 protein-protein interaction networks, signal transduction necworks, and taboie network, ach nctwork coctesponds to dillerent mode of interaction betwee biomolecules. Thus, one can think of the cell as made of several superimposed networks, oovith different colors of edges. Transcription networks correspond to one color of edges, protein-protein interactions toa dillerent color et Theresa separation of timescales between these ilferent network layers, Transcription networks are among the slowest ofthese networks, As we have disused they often show dynamics on the scale of hours, Other networks in the cel fonction on much faster time Scales. For example, signal transduction networks, which process information using inter ‘actions between signaling proteins, ean function onthe timescale of seconds to mites. We will describe some ofthe network motifs that appear in signa transduction net ‘works, sich as steuctures called multilayer percepts. Since we currently lack many ‘of the prevse biochornical details, we wll use toy models to wndestand the broad types of computations mile posibe by these motifs, We wil also examine composite motifs ade of two kind uf interactions (ovo diferent colors of edges). mn addition to these molecular networks, biological networks ean aio be defined on larger scales. For example, one may consider networks of interactions be ween cells. One Important celular network is the network of synaptic connections between neurons. We will examine the best-characterized neuronal network, from the worm Caznorhabuitis “elegans. this network: contains several steong network motifs. Notably, neuronal wiry shares some of the same network motif that are found in molecular inteeaction networks We shall discuss he possiblity that these motifs perform the same basi i formation pro- cessing fsnctions in both types of networks 6.2 NETWORK MOTIFS IN DEVELOPMENTAL TRANSCRIPTION NEIWORKS, The transcription networks we have studied so far are but to sense and respond to exter- nal changes. This type of network i termed a sensory transeription network. Sensory transcription networks are found in almost al ell, However, organisms also have another type of transcription nctwork, In all multi-cel: lular organisms and in: many microorganisms, ees andergo dilferentiaion processes they con change into other cell types. An important example is the development ofa mul- ticlled organism. Meltcelled organisms hegie hie asa single celle eg, which divides to ‘x the diverse cal! types of the body. As the cells divide they diferent nto different tissues. To become part ofa new tissue, the cell needs to express a specif et of proteins. "he spetic set of proteins expressed by the cell determines whether it wil Became, si, nerve or muscle, These differentiation processes are governed by transcristion networks, ‘known as developmental transcription networks (Davidson, 200); Davichon etal, 2002, ‘Albert and Othmer, 200%; Sanchez and Thilfy, 2003; Gunsahs el, 2005; Levine and Davidson, 2005; Stathopoulos and Levine, 2005), Developmental transcription networks of well-stadiod organisins such as frit fies, ‘worms, se9 urchins, and humans show several strong network motifs. They display most ofthe network moti that we have deserihed in sensory networks. For example, as se sory networks, the fee-forward loop (FF) isa strong network moti (Milo etal, 2004, dom, 2004; Pane et a, 2004 Boyer etal, 2005). The wast common FEL types in devel tental networks app neohorent FFLs, fst a6 he type-leuheren and type-t oO "into ey QI Ye Qo 6 OY apron cantor: FIGURE 6.1 Poe rancripton! back ops with two nde The double pstve ho hat tn t= ‘atom interactions andthe doue nepal oop mind of oer intratans Aolpepse ‘equated arson Hath ofthe frac ops as wa steady ster th Xen ¥ gone ON ot ah OF ‘hedoabe postive lop. and ose ON ene ther OFF the dole negine leap, in sensory networks (Chapter 4, Figure 4.4) Developmental networks also display prom ‘nent autoregulation motifs and SIMs. Tn addition to these motifs, developmental networks display 9 few additional network motifs that are not commonly found in sensory transerintion networks. We wil now desribe these network motifs and their function. 6.2.4 Two-Node Positive Feedback Loops for Decision Making Developmental networks display a network motifin which two transcription ftos regulate cach other. This mtual regulation forms a feedback lop, In developmental networks, the regulation signs ofthe two interactions usually lad to postive feedback loops (Figure 6.) “Thete are two types of postive feedback loops made of two transcription factors, Both types commonly appear in developmental networks. The fest typeof postive feedback Joup i made of to positive interactions, s0 thatthe two teanscription factors activate ‘ach other, The second type has two negative interactions, where the tw trnstiption factors repress eachother The double-positive feedback loop has two stable steady states (Thomas and DIA, 1990} In one stable state, genes X and Y are both ON. The two enhance each others’ production. Inthe other stable state, X and Y are both OFF. A sg- nal that causes protein X or Y tobe produced can irreversibly lock the sytem in st where both X and ¥ are ON and activate each other. This typeof bistable whch scaled 4 lock-on mechanism (Davidson et al, 2002}! Since X and Y are both ON or bth OFF ‘Recall dpe erence ack no «sae high eget A he dom ale ech be epee bp ease? Oe pre nnn ata he ede oie Fite on onion sera sprecble dy ante can er ot enon apt aii ‘Cotas othe eig eto fe kre Cfo)100 CHAP EER . PPM Go WIKI?) ae eck ps with toe 1) wth outoregultion ar conan oso atin deep lala setnork. 8) The 18 dnt per ‘tum 2 i set pang eam fates than the genes regulated by Y.! ‘ * Sy eng a cs l alte See tf tse tr ome Hore di ss dit si wh te ag aoe Jemongeot ot al, 2000), “eee hae ijt tothe hater," phage as Taf citne es enantio Insta hepa ot iat hee to mes. Toone mod, thee ode pew ‘ ‘nthe ictal genta fetter mead Xiah muds he ogc page 9A res eu DNA STOUR, Met ites ea abi at anemone e TCU 6.1 the gl edack tok mi it deveapmenta tarpon stake) Double psn Kes hp, When is acne X ad Y Begin be pce, They can remain locked OS Peer then Zasacttotetatines afr veri deed ie) (0 Daseepive feeb loop Here plated paced ahd 2 andthe eae oop {Facto sco te tte tealy Vs hal epee ARE Z ‘tseyred his ate an pra eten afer Zs deca Than bth cctv norera men 622 sys Fock an! Regolael [cedar ‘Two-node feedback loops can appear within lasger motifs in developmental networks. “Thete a7e two main ghree-node matife that contain feedback loops (Milo etal, 2008), ‘the first is a tlangle pattern in which the mutually regulating nodes X and Y both regulate ‘gone Z (Figure 6.20, called regulating feedback. “The regulating feedback network moi bas 10 possible sig combinations (combina- tions of positive and negative interactions on its four ees; Figure 6.2). 40 the simplest ‘case, X and ¥, which activate each other ina double-positive loop, have the same Fg Taton sgn on the target gene Z (bath postive or both negative. In contrast a double negative feedback locp will often have opposing rulaton signs for Z (Figure 6.2. The two sgn combinations shown in Figure 6. are coherent, i the sense that any two paths bperereon tv nodes have the sume overall sigo, Tiravlc tu the vogulating feedback moi, developmental networks show a network. motif in which a two-node feedback loop i ogulated by an upstream teanseription factor igure 63. This mis called regulated feedback. Again, several coherent sign combir rations are common found. For example, the input transcription factor can bean act. ‘ating regular that locks the system ON in the case of a double-posive Loop (Davidson tal, 2002) Th the case of a doulblestegative loop, the regulator can have different signs for the two foedback nes and act to wich the systemn fromm one steady state tothe other (Gardner et, 2000) ‘the seyulated feedback motif can be considered as a mermory clement the regulator sk lop from ane state to another, such that the state persists ve nee, the circuit ean remember whether 7. as active ean swith the feed aiker 2 i deactivatee {Figure 63) 0o HGR Taegan cases can eae dios the ler ote cl erin tine he oe ‘fsb prt ath epi hecsete cate prs nap sce oe shld athe ties Shown we nena whos easnd of pes inthe past. This memory is well-known feature of postive feedback loops (Demongeot ta, 2000; Smolen et, 2000 Xiong.and Ferrell, 2003) tean help celstomaintain their {ate even afer the original developmental signal that determined the fae have vanished 62.3 Lang Transcription ‘des and Developmental Timing, ‘An atonal importa anil atk mis in deveopenl ee hte sn sensor evors slang Nantrinalcaxaes, Transeo der ean etter n which ans for regulates, whine ele 2 ed son igure 6 Ase have seen in Ghats 2, he eapone ime of ech tages he ane go cone bythe degradation tion raft prten a tse othe ale Fs Jog(2)fa. Recall that for stable proteins, this response time is on the onter ofa cell gen- srt time eon 21)-Intresingy devel me oon ch he less hes of ane or ae cl eens This banc rare ged vith each si ivson sever ny athe cals Je fom Be see the ‘tre Hse tenses of rancrpn esas el ed ose dsl sei process Deslpment often employ seco epesurs ioe) whee ming poperesmay be mre ous with epee pean rection ‘aes than coeds actators (apport eal 200 eB an ., 24 tnteockedl Feedforward Loops in the f subtils Sporalation Network ‘The feed-forward loop (FFL) is another strong network motif in developmental networks |e developmental networks, the FFLs often form parts of larger and more complex cir sits than in sensory transcription networks, Can we stil understand the dynamics of such large cireits based on the behavior ofthe individual FFL? m . oi] > CURE 6.5 Th tanita negli eelpment ofthe els pore 2 Za epee [rps teu andres of gene Te ator lo made of pet incerent Ls which rer pulsed an Zand wo pr sobre F414, ane which peers a dlp ep of Zeon ekenrgr etal. 2006) “To address this quostion, we will discuss a well- mapped developmental network made of interlocking FFLs that governs difeentiation in a single-celled organism, the bscte- um Bacilus subs, When starved, B subtilis cells stop dividing and diferntiate into durable spores. The “spore contains many proteins that are nt found inthe growing bacterium, Iisa resting, ‘el, almost completly deydrated. I can survive fora longtime in a dormant state. When placed inthe right conditions, he spare converts itself agnin into a normal bacteriue. ‘When 8. subtilis makes a spore, it must switch from making one subset of proteins to making another subset, Thi proces, termed sporulation, involves hundreds of genes. “These genes are turned ON and OFF in a series of temporal waves, each carrying out spe cif siages in the formation ofthe spore. The network that regulates sporulation (Eile berger et al, 2004) is made of several transcription factors arranged in linked coherent and incoherent type-1 FFL (Figure 6.5) “To initiate the sporulation process, «starvation signal S, activates X,. Tis transce tion factor acts in an incoherent typecl FPL (I-FPL) ta control a set of genes Z. In this ILBFY. X, directly activates Z, and also activates Y, which represses 7, The ILL gen- erates pulse ofZ expression fs described in Section 4.7). A second FFL is formed by Y, and X, which are both needed to activate X, resulting in a coherent type-t FEL (CL-FFL) swith AND log, The CL-FFL ensures that X; is not activated unless the 5, signal is prs ten (@s was discussed in Section 4.6) Next, X, ats in an J1-FFL, where it activates genes Z, aswell as their repressor V,. This results in a pulse of Z, genes, timed ata delay relativetw the frst pulse Finally, ¥, and X, togter join in an AND gate CL-NH, go activate genes which are tendon ast. The result isa te-wave Letnporal pater sta pulse of ‘expression, fllowea bya pulse of 7, expression, followed by expression ofthe ate gees Z nce the FELs in this eloors are combined iv a vay that ullizes their delay and Dolke-generating features to generale a temporal programy of gene expression, The exe ede! ITs generate tw pulbes of genes followed by a third wave of late gens (Figute 6.5). be PPLs contelling ZZ and Zane vetvally multioutput PELs because Zp Za and 2, each nepeesent large geaups of ones, "Ihis design eau generate finer temporal pro rams within each yeoup af genes as describ sm Section 5.5, We see thatthe FL i this network are linked such that the dynamics ofthe network can be easly understond based on the dynamics of each FEL, A similar situation was found in sensory transription networks, ia whieh BFLs are combined ia particular fash- fons to farm multi-output HLS. The dynamics of malt-output PELs can also be waver stood hased on the dynantics ofeach ofthe eonsiiuent theee-node Fs. Tris important to mote that there are, in piaciple, many other ways of Hnking [¥Is. Most combinations ‘of inked FFs do not lend themselves fo easy interpretation (Can you draw afew ofthese possible configurations) In short, the FEL in the B.subts sporulation network al in sensory netwotks yee be linked in ways tht allow easy interpretation based on the {dyramsics of each FFL, in isolation. This appears co be the case also for network motifs in many other developmental netiwocks Such understandability of circuit patterns fn teres of simpler subcircuits could not e oF have evolved to make life casicr for biologists. Understandably i a central Feat ‘pincering, because engincers bull complex systems out of simpler subsystems that ave well unxlerstood. these subsystems are connected so that each subsystem retains its buchavior and works relay. His an interesting question whether understanndabilty might bea common feature of networks that evolve to function. ob NETWORK MOTIES IN SIGINAL TRANSDUC HON NETWORKS: We have discussed transcription networks that operate slowly ona timescale that can be asslowe asthe cel’ generation time. To elct rapid responses, the cll also cuntins mut faster information processing networks, called signal transduction networks. Signal transdaction networks are compose af interactions between signaling proteins “their fnnctn ic to sense information Leotn the snvieunment, proces thi iormation, «accordingly regulate the activity of transcription Factors of other effector proteins. For example, cells in animals usually do wot divide unless they are stimulated by ho. ‘mine proteins called growth factors. Specific growth factors can be sensed by eel, trig ‘ing signal transduction pathoray that culminates in the activation of genes that lead te call dvisio ‘The inputs co signal transduction networks are typically detected by receptor proteins (Figure 6.6). Receptors usually have one end outside of the cell's membrane andthe other cod within the cell's cytoplasm. Their extracellular side can detect specific molecules called ligands. Binding of the ligand molecules causes a conformational change in the receptor, causing its intracel sd to become ative and catalyze a specific chemical NV vn oh ct Teomerpin fee LIGURE i Prtein kn ata gad bos he epoch ads. sly through apr jo ‘eas phospoclain of kinase X. Kinase Xisactte when pnaphoeysa, 39. The seve Ks Kp lespboryne start Kine, YT aetie kina, pia ar, phophorltes ZT a Re he scab, 2p, poghoriate tarcron factor F, making at, Rolly enters the mae ed ‘tats representing, Phosphate ermove he posta groups gh arro) modliication to a fusible messenger protein within the cell This ification can be ‘thought of as passirg one bit of information from the receptor to the messenger. (Once modified, she messenger protein can ital! modify eecond type of mcseengor protein, and so on. This network of modification interactions often functions on the tim scale of seconds coraiautes,Itofien culminates in modification of specific tanscription Factors causing them to become active and contral the expression of target genes, The structure of tignaling networks sa subject of current research, and many interac tings areas yet unkown, Furthermore, as we will seein Chapters 7 to, the precise func. tion of these networks can depend on subtle biochemical details As a result, we ca at present only draw tentative coaclusions about the structure and fenetion of signaling nt: ‘works, Hovteve, th available data alteady show several intriguing network motifs Here ‘ve wll focus ona neti made of interacting signaling pahays that appears throughout diverse signal transduction networks, but dees not appear in transcription networks.106 = CHAPTER 6 ee f T J ‘Ww 6% ‘an he an Generali ofthe dann ate bain by duping ef the fn ee it all of itcedges Tate enerazton rlalss nt ein pal esac er GA INFORMATION PROCESSING USING MULTEL AYER PERCEP TRONS In signal transduction networks, the nodes represent signaling proteins and the edges are directed interactions, sach as covalent modiiation of one protein by another. Signal ret works show two strong four-node motifs, the bi-fan and the diamond (Figure 6:7) erkovita et a, 2005; Mcyayan etal, 2005b). The bi-fan is also found ia transcription reworks, We saw thal the bi-fim in transcription networks generalized to single-layer pattems called dense overlapping regulens, or DORs (Chapter 5, Figure 512 and Figure 513) The diamond, however, isa new network motif that is not commonly found in tran- scription networks, ‘The diamond network moti in signaling networks generalizes to form multi-layer pat terns (Figure 6.7). These paterns resemble DOR.like structures arranged in cascade, with each DOR receiving inpats Irom an upstream DOR. In contrast, in transcription networks, as described in Chapter 5, DOR patterns de not oceur in cascades; that Is, 2 DOR is not normally found at the output of another DOR, The multi-layer patterns in signaling networks usually show connections mainly from ‘one ayer to the next, and not, say, connections to nades that are two layers down. Such structures are similar to patterns studied inthe flelds of artificial intelligence and arti al neural networks, called multilayer percepteons (Hertz eta, 1991; Bray, 1995), 64.1 Tuy Mode! for Protein Kinase Poreeptons [et us analyze the information processing capabilities of these multi-layer perceptron ‘We will use « toy model of a common signal transduction modal, protein kinase cas- ‘eades (Figure 66) (Wiley tal, 2003; Hoenberg etal, 2005; Maayan et a, 2005b; Kole 11, 2005), Protein kinase cascades are information processing pathways found in most HOUR. (8. Doobephosphorltion in protein kinase asad: pon Rinse X,Y Ze omaly | Phasporyaed a a ste end le eqn eth papular ll ety. eukaryotic organisms! These cascades ae mule of kinases, proteins that catalyze the phosphorylation of specific target proteins (phosphorylation i the addition ofa charged PO, group to a pec site on te target protein). The eascade i activated when a recep tor binds ligand and activates the first kinase inthe cascade, X. Kinase X, when ati ‘ated bythe receptor, phosphorylates kinase Yon two specifi sites Figure G8), Kina ¥, when doubly phosphorylated, goes on to phosphorylate kinase Z. When kinase Zs dou ‘iy phosphorylated it phosphorylates a transcription factor, lading to gene expression. Specific proven enzymes called phosphatases continually dophosphocpat the kinases (by zemoving the phosphoryl group) Therefore active prose Kinase cascades display a cycle of phosphorylation and dephosphor ylation. Protein kinase cascades often use scaffold proteins to hold kinases in clase proximity, (evctenko ea 2000; Parke al, 2003). Adaptor proteins can connects given carcade to diferent input receptors in diferent cll ypes (Pawson and Seot, 1997). Thos, these ‘aseades act a5 reusable modules (Schaefer and Weber, 1999 Wilkins, 200} the same ‘cascade transuces dierent sigal in diferent tisses in a organism, Protein kinase cascades ate usually made of layer (igure 69), fen thee ayers In Ue fest layer, several related kinases X, Xp... can activate the nowt layer of kinases Y, Yq These, in turn, can activate the third layer of kinases Zy Zaye Tis forms a mult dnyer perceptron that ean integrate inputs fom several ecepor igure 69). tan ewok 3 pti or ina us roi open Gr sg rans. However: he pl ln bac ny sip than he pen Kn anno ears: Baie. ‘et stn waa tee tha ass ose! ry tn aise sponte el ‘ane het ad Che the eons sem dered Chapter). mci srr alin ets ‘eels is psp ey weer Intersting oc ny cle sigs eh er an ‘eho gating nr108 mw CHAPTER G4 7 Mtge gersptons in protein Kinase esd Soe sent cea the same el ‘inact psc op hye ian expose ona Lab yr hc sca is a ‘ins enc fa cunpaphonyatena ofthe hans a thee ee ‘To study such multclayer perceptrons, we will write down a toy model forthe dynam ies of protein kinase networks. "Ihe gol is to understard the essential principles, not to develop a detailed model ofthe system. Hence, we will use the simplest kinetics for the Kinases first-order kinetics (see Apperlix A.7). Firstonder kinetics means thatthe rate of phosphorylation of Y by X is proportional tothe concentration of ative X times the concentration ofits substrate, unphosphorylated Y, denoted Y. sate f phosphorylation =v XY, ‘The rate of phoxphorylation is governed by the rate v of kinase X, equal tothe number of phosphorylations per unit time per unit of kinase To begin inagine » kinase Y that is phosphorylated by two diferent input kinases, X, anal X, (Figure 610) The phosphorylated foray of Y is denoted Yq an the unphos Phorylated form is denoted Y,, ‘the total number of unphosphorylated and phosphory- lated forms of ¥ is conserved yrye¥ (oa "he rate of change of ¥, concentration is given by the diferece between ts phosphoryla ‘oso inpot kinases and the dephosphorylation of Y, by phosphatase at eat at ¥ ca change du to teamsrin afte pene: Thea change at ely ch aleat Ie phspteiion-Jeonghaeiain rca Hence te aa on ion ce be esl cnso we the inal a eben pin ee Ne a eh pocins aang coca are esctinally pt hyn consi sms eee mY 1 WY. hae 4 Summa pein W488 FIGURE 1. Fraci of actte Ya Gin of he wg um he ipa hse cit y+ ssn the moda wth ean neter Showa eat curve Sean dub sph [ti of the weighs and wae the aise be int kan vats an the apts ae AYALA WX YA, aay, a2) ‘We wll tudy the steady-state behavior ofthese cascades (hough Aynamical functions of kinase cascades are very important se solved exercises 6.4 and 6 5). At steady state (lt 0), these equations ead toa simple solution. The faction of phosporyated Y isa fn don of the weighted sum of the concentration of active X, and, with weights, and YA = FX, #04, XB) (64.3) ‘where the function fin Equation 64.3 isan increasing, rating Function (Figure 6.10} Wi, a9 ‘The weight of each input corresponds tothe rate of the kinase divided by the rate of the phosphatase: wewla and = wa 49) Jn other words, the concentatio of poplar ylaued an hcrensing funclon of she weighted sum of theo input kinase activities! When ¥ is kinass that needs tobe phosphorylated on two sites tobe active, the input furction Fis even steepce (Fig 6.10; solved exercise 6.2) te) 46) “These shaped input functions lead tsigfeat activation oY only when the weighted ‘sum ofthe two inputs is greater than a threshold value, which is approximately: that ogling prince apy ove ssa inp sony ie ‘evs pete aad Li 208 Pa 200, Be110 CHAPTER & SANE 118 Sin aye pve op ones Ne he nats X aX tengo handy te ithe Seed gift er hon Soetrkos rose ite porte nl ean te ad ge i ‘etiam gue 2 a ge 61, the age of ties of Xan bats nd An ati of ria ftir kestrel portrayed nthe ges Ww XA WX (an sox codeine ts of atin y= Thre 1 yb ste stele ts eo roa sept igh cD ‘hse on en tht ie a he ry fe an sgunigetonem mann ewer on gen Thigh cps nig gay ah ad ae Ginn oon A gi tn te eae he Hen cin pen gh cys ot OR ue eps hac én nly carpe we win a cn on mae eg yee mechan a wictioe tp aes ny rth pa es ngs 8 ‘Sfiutantbterifanonecitinn Sins onatet Tie shmatsiny fc tet s t oghoon e on Sweat nso py edi ie oi the ee he iter el ton ema NEIWORK MOLES @ att the plane ofthe two input activities. More generally similar functions describe a ayer of Input kinases Xy Xs Kye which regulates a layer of outpats Fy Yorn Yo The actvily of Y, Sa threshol-like function of weighted sum of imps fy y+ Wya Keto + ¥y 3) ‘Note that ech, sits own set of weights correponclng toitsafiniies to the input kinase. ‘These single-layer pereeptrons can compute relatively simple function they produce output ‘ifthe summed weight ofthe inputs exceeds the threshold This threshold of activity can be represented asa hyperplane in the n-dimensional space of input activities Xy, Xn Xe We will continue to use just two inputs fr clarity though Ube conclusions are vali for percep ‘rons of anys, 2. Multe-Layer Peeeptonts Can Verfonn Det ei Conyaaatine In our toy model, a single-layered percepiton divides the X,-X; plane into two regions, ‘separated by a straight line. This allows computations af functions similar to AND gates and OR gates, We will now see that adding additional percepteon layers ean allow more intricate computations. Consider the two-layered perceptcons in igure 6.12. Each ofthe kinases inthe middle layer, ¥, and Y,, has its own se of weights forthe two inputs, X, and, Therelore, each has ifs own input function in whic a straight line bisects the plane it a cegion of fw activity and a rogion of high ativity ‘The two kinases Y, and ¥, can phosphorylate the output kinase Z. These kinases only PhospnoryateZ when they themselves are phosphorylated (ecll that protein kinases are ‘only active when they themselves are phosphorylate). Thu, the phosphorylation of Z is 4 weighted sum of is two inputs, withthe same function fas before (Equation 6.44 oF 6.4.6) and with weights w, and w, 22: {thoth weights and ware small bot, and Y have tobe phosphorylated to cross the activation tneshol. This mens that Z is phosphorylated ony inthe region of XX, plane, where both Y, and Y re seve. Th eon is defined by the intersection the tno atvity regions of Yad Ys, This terscton results inactivation of in 8 "egon of inp space whose boundary Is defined by two lines (Figure 6.128) In contrast, es above that single ayer perceptron allowed tpt repons defined mor coarsely by single straight ine, Henes, the ditional perceptron ayer affords a somewhat mre ‘ined activation fmetion fv. My wa YD (as) Adhtional activation fonctions ate shown in Figure 61. Here, the ction egion Fe union ofthe a atvtion replons oY, and Y, because the weights, We ste age enough so tat ithe inp Kinase scent to ative 2). Again the ative "on region isbn hy tinea segments. An ational level of deta an be ged whoa the mide layer contains a speie Phosphatase instead of kinase. In this cine, the phosphatase remmes the phosphor!ouge 2.5 verano wren cAMP) rei AMP att= emasoornonetance [nie ) FIGURE 5.4 Temporal order i be agiszebceetue pte, The poses eaten deine ‘order wih dls mins teween promt. Color br stom expres fm te peer a he i Ferenopeons inte nysem, ensued by meas of nieacen reporter gene, The postion of eich ene reuctin the pathway hat re apiin sow, En Zar 2004) Time [min] FIGURE 5.9 Tanger ok i the gel sper fF, coll, Coad hae ae the nommalize eaesson ‘ech prmoe where i ab ee ih ean. Aso ech feet a ie teas green escent (GAP reporter. The peat nds mulches the aso one oe Roel, ‘ewe prin ae aed gang the nacelle eter ten CPi Kite 200),NEIWOKK stosHS mang Imoxiication and slectivey has « negative weight! A noyative wig intecatly shape activation regions for Z, An example show is setvated when the inpals X, so. this design leads toa wed sa lea «tare igure 6.18, in whic Mt X, are such that node ¥, is activated ut nade Yi ike rion of 7 activation, Other weights can led ton activation of when either X, ane X, are ative, fut wot ft ils to an ekcasive-oy on XOR gate Figure 13h). These types af computations are nat possible wt a perceptron (Hertz tab, 1990, pte ttn aon 0 mabe ‘Aad additional layers ean produce even more dette [ut activation region is formed by the htersectios of te different weighs ofthe perceptron, Uierent regens dias by 2n summary. mhislayer perceptmoas can pertaran more detailed! competitions than single-layer perceptrons. Our toy model considered cach kinase asa very simple unit that sens inputs and activates downstream targets whew the sun exceeds a threshold. Mult layet perceptron allow even such simple units to perform arbitrarity complex computa. ‘ons, base on thepower of combinatorial layered information processing (tay, 1993). ‘Mul-layer pereeprons sinitar tothe ones we have discussed have been studied in the contest of artificial intelligences. They were found to display several properties tht may be usc for signal transdution in cells (Bray. 1995). {wil now describe thee of these Dropertis very billy; more details ean be found in tests suc as Herts et al (1990, Malt-layer perceptons can sbaw discrimination ration, and graceful degrada tion, Discrimination is the ability accurately recognize certain sini paler, A ‘ulttayer pereeption can be designed with weights and connections so that ca tell the dtference between ast of very stir stinul patterns. Generalization isthe ability ‘oil n the gaps in patil stu pattern, A mule-ayer perceptron ean be designe! ‘with weights nd connections so that it ean eespondl iflerenty toa seta illeeat sina paerns If present with » slinalus that only partially resembles one of the orginal stint he circuit wil ct ast saw te entire input patern, Finsly graceful degrada tion seers tothe fact thot damage 1 clements ofthe perceptron or is connections docs ‘not bring che netwerk to a erashing hat Inscad, the performance ofthe netvark dete rats, a levet proportional tothe amount of damage. tice, notations i 4 signal lent soritimes have only small elects ex cell respanses, pattic 2 these thre phen ieput FFLs Sunes se spt SIM, Bh are fe sgn ome peer) {Bf donee cote BOA) TD eget (9 come teenie 1B meri echo oe en pase rnsrtonatwork: lips represeat ansripo itr tht rea he sigan rom be eoionrent Ws Tangles ae ou of age nena, generalization, discrimination, and tne Xa hina fel the ing ten Mah =, 5,08 -Yo op Sao Xe) te eh Tatkehnale (A agave wg ery wt on sh Speninanan Toei eceoacen egy Becerra sn ns its oe pote srl ec an see oct Sp sts re a {ret he eget. Om nhc ny heater gta oer seine al wantin ved itn caput ha co atl sen nat mae ann, tr ke (have fwchuy seme ct redone ays cron FIGURE 5:14°The gba strstr ofthe clA14 CHAPTER & REIWOKK MOINS 115 {raceful degradation, might characterize the functioning of signal transduction networks incall In summary, multilayer pesceptrons allow even relatively simple units to perform. detailed computations in response to mulipte inputs. The deeper ane go ‘of the perceptron, the more intecae the eomputation ean become, othe lyers 6.5 COMPOSITE NETWORK MOTIFS: NEGALIVE __FHFDBACK AND) OSCILLATOR MOTIFS . We have so far discussed protein signaling networks and transcription networks sepa "ately In the el both networks operate ina integrate fishion. For example, the tpt ‘afin rrnedvotion pathways i often a teenseeiption fot ‘Because transeription and protein interaction networks function tether, they ean be escribed a a joint network with two coloes of edges one coloe represents Uascription| Interactions, and a second color represents protein-protein interactions (Yeger-Lotem et al, 2004), In this section, we will briefly mention some ofthe network motifs that occur in such two-color networks. Network motifs cam alsa be found in networks that integrate ‘more than two levels of interactions (Zhang eta, 2005; Ptaceket al, 2008). ‘An example of a two-color motifs the following three-node pattern: teanseription| Spt 2 hat ated when ether ce Reis es Ze whereas Y hte Ty Ze i i i 324 factor X transcriptional regulates two genes ¥ and Z whose protein products retiy Bek | interact, for example ¥ phosphorylating 2. (Figtee 6.143). This reflects the fact that tran aa scription factors often coregulate proteins tha funtion together. $22E ‘A very common composite motif fsdback oop made of two proteins that interact Hor ‘ith each other using two coors of edges (Figure 6b)! tn this mat protein X ia tra Ege | scription factor that activates the transcription of gene Y, The protein product ¥ interacts FEES | sthoton the posi el (ot rnserioniy) afer ine remanent coos eet tive regulation can rake several forms. In some ces, ¥ enbanees the ate of degradation paad of protein X. Im other cases, Y binds X and inhibits it aetivity as a transcription factor by EES 2 | __ preventing itsaces tothe DNA, This typeof feck occurs in most own gene sj BEE, | temfrombacterinco humans (ha et ae 200 bey ‘This two protein neptive feedback moti 3 hybrid of two types of iterations: One 22 | interaction is txasciptonal in hich Xsctivates¥ ona sow tvnecale the other iter ae Btn ecu an the prt eve in wich Yin X ahs ep messes eyg2 be noted that purely transcriptional negative feedback loops are relatively rare (develop: Sase | Mental transcription networks usually display positive transcriptional feedback loops, as BEES | cused above). Im ether words i rare fr ¥ to repress X on the tanseripion lve Be2e | What could be the reason that composite negative feedbacks are much more common than parely transcriptional ones? || Reno east octet hs ng ee ken tbe etmek Merete poe Paar pthwa eh tens n he py Theta cs | Rese digo ons sseond saat The sow sn te fan ehanpr the patentee ‘edeir annoy nprir te pect ss sel ne fem enone | inte ities cn ol uy rapesa CHAPLL 16 2 serena) pet GUN 614 Composite sta nt ae of ane is r an sig i ro dc (rin for Xe eo ge a 7 won procs itrct ( &tr eck lo with ene traactipton am lon ro 98 osteo tists rg To nerstaed composite feedbick, we can turn to engineering conta theory Feed back wha somone ulated conn wed gs g- Apical us ofthis type of eedbac sto sablize a system. For exampe, heater thats Smits heats om scam ns eae re op yc inet hens ge 1 The Hert comptes he deed empresa temporatre and adjusts the power accordingly: ifthe temperature is too high he powe ‘ofthe ester sre se that te room coos down. Afr sae tne the temperate stabilizes around the desired temperature (Figure 6.16) One ean for wing two ineseles fast then te enhance tality he pel eepnee fine a he thers totes talon te coo tmp a vo en me a vat of rey ta takes 15 mi o eespon to temperature changes, the eater wo sce feck ed on te aly tant pa an emperre wos ct soaogy. negate edt on ade ft slow eosin net is more prone to fasta thon a fnback loop whee fast interaction contra slow an. Fe Iocelet macnn we msaheaa etm of sy se Sos 0) Wess saity around he sat so cle hme) i desire mar be sens ater hp sens ply ste leet ehvor — aoe ain Win, 20 Gober 208 Mara, 208 The elec wish calls periodically duplicate their genomes and dive, i an important opilator ( fom sve late) in this eee. ners thatthe cn NEAWORE MONS 447 [ome HCL 6s Neate dc gssng. fl tal oh dees A he snd ya her tina sted tern seal ematical the pone ote eter tse and power inurl eprint ree ey mich ne ines tha the eter talaer oul tre edo paneleeaanee as tds emperatr ctl nde pl ots ged) tal 2002 Ante well oem example the cradin clack, enarhablyacceate biochem crit that produces oilstions on the sea one day. rod whichrcon te ¢eteined to flow it vations in signals ich as fenperatute an ight Other se lator occur i egelitory systems, sch as tanscriton Lactrs who concentration sty ose in sponse specifi sige Hotinan ea 2002 bons 20s, Late a 01 No a, 2004, Osillsiors are sho foun in eating het ale. ping ures, an devapmnil processes tht gens repeating modus ties orgs and Gots, 200 cogil osiaos have wiypisl carat: thee Ung sway spiny snore precise than tir ample (lahat al, 2004 Ahaleeew ea 200% Data Pulses inthe osiatnn acura ther accurate tne intervals bot with yng ang des. The source ofthe variation in th ampli apeats tbe slowly acy teal ‘sin protein prodection ates tha inherent in bochoialeneotey(Appenoe oh Many blloialeilitns appear tbe siplementad by a two-car netourk. ot tesa embeded in crus ote interactions This moll 9 compose rege feedback opin which the aos factor % also dle pontine sonegetaion euce 6.179) Oomenning etal 2008 Tn cal 009) thacheotean pene ey ons wth obs ning dept uctustions i the big pcancter ofthe compe eos (Baka sn Le, 200; Vile, 200% Atkinson tal 2003) Themechanions ot Tabs silaton ised on hysteresis the amis X aunt bythe area ‘er lop. this csi blogs 0 fay of models known an eon cannes ‘Actics possible desig for aslo cre i eback oop nad of mip et |e hoked up inal to form a negative fsck loop. For tame thrce prenos booked up in a cycle are called a repressilator (Elowitz and Leiber, 2000) (Figure 6.17, {eee 49). This ict belongs tothe family of delay omltrsa fry that ete ene Se show noisier silitons with es prec ting Extensive thar werk on by ‘lowilltrs an be and inthe Further reading section attends stamtee,118 aw CHAPTLS 6 LA te Neate etka homed tn doa pe melas th reso ceo nr we la. Gener he org the ition nee "rete nodes n lation the sdarpng fret on ech noe ee 5 dered rte he higher the tendency lr selatins 6.6 __ NETWORK MOTIIS IN THE NFURONAL NETWORK OF ©, FLEGANS sof science deal with interactions, including sociogy (Helland ‘Many fils of sence deal with networks of interactions, including and Leinhardt, 1975; Wasserman and Faust, 1994), neurobiology. engineering, and ecol ‘ogy, Network motifs can be sought in networks from these fields by comparing them to sandomized networks. One Finds that |, Most ceal-world networks contain a small st of network motifs 2. The motifs in diffrent types of network are generally different. re the motifein networks ofiifferent sizes. ive statistical sgoifcare Pranplescan ean in gine 68. Tos tnecin dine groph profs profits py thee Sfeach pect bap oto randomised nets gure) Ths co oie ety re ifr ens Teme scone eine tach peor perl ferent Howe hte Stew ising cans whch ured nets hare sm tor i “rant ptr tha re er tan aan). Ti eco ea whee sping the eon nero he nate sage ty wae compose CFs 10 ee igure 620, Theayopicmmetns teen lh 20 neronsin ns again were manos by Whe, Beer std Cegae Chit aly 198) The wing ds te 07 “Totaly Hom inst nhc meer thee 92" tran ie X > ¥en yeron Xho ype conoeton 8 & Coe) Le \ '@ etarmit n GUE 617) network si found in mn isos wtiv edhe op ana posit location a this tl aetven Yo slow ee snd ethos ie Y ints Xa rapstimesale (Arent mae hee eel ake ntti the epresattr aly sons masy cain in he resent a atone pak tion aero he pti, lor stems composed of somos neuron ¥, Thus, this network can be searched for network motifs made of neurons Seth ingly, the neuronal network of C:elegens was fund to share many of the motifs found in anscription and signal transduction networks, The network motifs in the C.efgans neuronal network are similar to those fond in biochemical interaction networks, despite the fact that these networks operate on very «lierent spatial and temporal scales. The scale of the neuronel network is that of cells, and the response times ate milliseconds, The scale of transcription networks is that of nanomeer-size biomolecules within a cell, and the timescale i minutes to hours. Yet ‘many ofthe motifsin these networks are similar. Por example, the most significant three ‘ode motif in the newsonal network isthe feed-forward loop. An example of neuronal feed-forward loopsis shown in Figure 6.21. Why is the FFL 2 motif in both this neuronal network and transcription networks? One point of view is that this is a coincidence, and different histories gave rise to simi far motifs! We for a different view, thatthe similarity in network motifs reflets the ‘ct that both networks evolved toward a similar goal: they perform information process ‘ng on nosy signals using noisy components, Both networks need to convey information between sensory components that receive the signals and motor components that gen- tate the responses. Neurons process information between sensory neurons and MOLoe ‘neurons. Transcription networks process information between transcription factors that ‘eceive signals from the external world and structural genes that act onthe inner of outer ‘evironment of the cell, This similarity i the function of the two types of networks raises the possibilty that evolution may have converged on similar circuits in both networks {0 perform important signal processing fonctions, such as sign-sensitive filtering, This he pia angular pe al he FL. might aed fo the spit os. Netens ht ig tend cnet rte ae ham data eos ech {boon fee eampnauce wine shoe putea, besane PH Sehoe to and ee oe a ‘esas cl fo nwa whe seer woul rr le ordape,Hao wed roche ‘fea pe ais X, and Zameconnved acylase any aber pers kes elon, 2961 ed ecko Pw eto ceany in tna eth tee Bieta, Mos ahe ine Ts ond ber mts Me Seanad nes ry Botha ereHG ttt Network mie unin Wolo lopkalnetwor The mes ale. ‘ge foc et ae swe, Fresh ti the saset of appeances inal otk ed nis therardomired newoeks Nye ll lus manda are sam. Avo gute eneat Tia agmivanethe sone = (Nyy Ne_tieshowen NS, ot spaiean Shes ieee teat dst ine nith comply de its aes Tbe ntworksimesy ic meceioe nore rn in apm ling cura contstedb leat ve synaptic itersnns eee ‘lf webs, eset pec nthe hte Rk Lab) bt tracts ton “aD icon Ra Y te vz ace shown. Crom Milo al 2002) : My Compete at [oo aster ss | ad tanean 2 i See ete ca ef a ce fg i ee, me Me iste? 85 wets Bas ae A i Tisica a re aon (crevcgeti) 4 moe] 7 boot oF tesa yo Mf Re rccatcea| se eM ae ey os [we tet nw | aw <7 ce /N\ x | DTD REG ag age - oe Subgraple ou mu » | ACRE Set poles de sods pana ae he pre ig ae wa i esc nat aps kce ond gph upans pester tee ao aa : eee ee tem ronclastateg sed onvonterecctiig eee . ‘teal lace es (Wo Ws Wes ai) Theyeab ote nels ee ee Wives Sask Wes erent hes legons sheeo sender ke cee 7 \ ee lagage neterks ve wl es and ees eon rs that le ll ech eae a ‘aeeaterae eas ening opi ween wth se ase meen ncHGR 1.20 Map of symaei nections Between Clon nos. Shown any cn netons bw ncuons it the weet hel fra Darn, PD tea wena) hypothesis can in principle be tested by experiments onthe dynamical furtions of neural K)-aY 5 ere ais the relaxation rate related to the leakage of current through the neuron cell ‘membrane! and the weights w, and w, correspond to the strengths ofthe synaptic con- nections from input neurons X, and X, 10 ¥. ‘As we have scen fr signaling cascades, these weighted surns can generate either AND ‘FOR gates (eee Figure 6.1), For example, if both weights at lage, so that each weight times the maximal input activity exceeds the activation thresholt (4, Xin» Ky anid Wy Xsue 2K) the reslt i an OR gate Because either input ean eause ¥ tobe activated. An Tein caret sen with este eager C.-C124m CHAPLER ae aa 7 ‘tinal 3) wo np EFL, he np unctions nd activation thresholds te shows.) Dynamics of he and AND uate occurs ifucther weight Is lage enough to activate the neunoa With oly one Input, so that both inputs are needed for activation, With these equatens, we can conser the two-inpul FEL network wot (Figure 6.229). 1m this cireuit, neuro ¥ compares the weigited sum ofthe inputs fen und X, toa theestld. ‘the neuron 7. is alse controlled by X, aad Ny, and ination receives inpats fs nero AHL = BOG KWL WY KZ (66.2) Experiments on the nose-souch system of Fiore 6.21 suggest that 7: can be activated by the fllosng loge input Function: (X, OR X,) AND ¥ (Chatfie et a. 983). In other words iter of the two inputs X, oe X, covided that ¥ is also active (Pig: tee 6.222) In this case the response to a pulse of input signal from either input ean be «ssily understood based on Ue function af the simp, thees-nod coherent FF. Each of the two TFT acts asa persistence detector with respect 10 ite stimulus. Fence, the out pat neuron Z can be activated by either inp, ba oly the iuput is persistent enough ‘his persistence detection occues because the voltage af ¥ must accumulate and cross its sctivation threshold Ly activate ZA transient activation docs net give Y sufi time to accumulate, Therefore, transient inputs do nt cause aeivation (Fig 6.22), 1 addition o its function 96a persistence detector, the mult input FL ean perform, soincidence detection of bret input signals: a short pulse of X, activation, which by itself ‘snot suiient to act vate Z, can still do so i there sa shoct input OF, i chose proxinn as demonstrated in Figure 6.22c. Thus, a transient input can cause wetvation i iis ‘ollowed closely enough by a second teansient input from the other input neuron. This ‘incidence deeetion function can ensuge that a short pulse can activate the system jor ide hat it has support from an additional inpet at about the same tnt. "This feature is ‘ade possible bcauss Y acts 36 a transient memory, storing information fr timescale ‘of ia (ou the ecdr 0f1 ms) The timescales ofthe dynamics are determined by the sesponse tives of the neuron Yoltayes. T= log(2¥a. Whereas in transcription cits Ty» often has timescales of hours, in he neuron: circuits fC: elegans, T,, has tnesciles of tens of milliseconds Despite the vast ditference in temporal and spatial scales, the same simple mathemiti- ‘al reasoning can be applied to understand, at ast appruximately, the dynaies of net wor mot in boll networks. 66.2 _MuitsLayer Ferexptnons inthe Cela Nerul Netwrk When examining patterns with four or more nodes, ane finds that the most abundant etwork motifs in the synaptic wiring of ©. elegans are multi-layer perceptrons (Figuce {623}, These motifs ae similar wo those we have seen in signal transhiction networks (Sec- Won 6.4) The main structural difference is that C. elegans ul-lyer perceptrons have a Fisher abuadance uf matual coancetins (feedback loops) between pairs of nodes in the same layerVY & Coane Goomezen Cramezote CxoRt ie © % @ @ Bs A Re ma A YR HGMRE 621 Fe and sade sto mn the euro c wn nthe era ewok of ig te he "errno nih tlio wi eh meter Ci the cancer of the sabgaph mated by 10% where concvtatin deed the Ir of appearances ofthe sera dd by he lal umber of apperscs ef apap of he Same nth nt he ube tnd des ht aceon dn close dpe sore hte te sn -slgoriti suitable far large subgraphs and large networks. (From Kastan eta, 2004.) * son ae ti he antn of hese mul cesing ofthe tp hat ne esi in Seton 6 Anas eth pete compton peroed by nero perception cuts il depend on cere messeent e Set cath eg oi sant Te rei cl be eden oe iran tess yey posses within she naron andy ing oles aie tn os We laste ned ‘worse sires in th operation : ns canmevesiog uct | ies sears tes ofc, em yl tn, yng et tin hel tn te fal th euro va sophie el seo pom compton doops ine. The prose hcsion oer oly thesinpes eer r tse trot Final aaj ofthe state of euro nets of hgh eget 3 ts cate Hm pet at se esha la Zion. Pliny stant the xs network ts othe te elo ini Ganga, 205 ann eto cnn oh bin fat ene nd et 0 Saale 7 SUMMARY oo . ‘We have scen in this chapter that each type of biological network is bik ofa distinctive set of network motifs, ach of the network motifs can carry out defined dynamical Fane- tions. Developmental (ranseription networks display many of the motifs found in sen- ory transeription networks. Tey aay have adlitional network motifs that correspond 10 the irteversihie decisions and slower dynamics of developmental process. In particular, towo.node postive Feedback loops, regulated by ¢ Uhied transcription factor, can provide Tock-on ton cell ateor provide toggle switches between two diferent fates. Lang cascades can orchesteate developmental programs that take place over multiple cell generations Signal transduction networks show faster dynamiss and may ailize multFlayer per- ceplvons to perform computations on numerous input stil, Mult-lyer perceptrons fan allow even eelatively simple vnts ( perform detailed computations, Multsyer pereeptrons can perform more ftrieate computations than single-layer pereeptrons ‘The dynamics! features ofthese networks are affected by feedback loops and additonal interactions, We are only beginning to understand the computational funetions of signal transduction networks. Integrated networks made of eilferent types of interactions can show composite net- ‘work motifs, A common motif ts 2 negative fedback Toop made of a slow transerption Jeraction anda faster peotcn-level interaction This feedback loop can generate robust ‘oselltions when coupled toa second postive autoregulation loop. ‘We have also examined the network of synaptic connections between neurons in the ‘woent Again, we saw thatthe patterns found in the neuronal network are ony a tiny fraction ofall of the possfble poterns of the same size, Hence this neuronal network hava sivuctural simplicity reminiscent of that found in the biomolecular networks we have studied. Moreover, many ofthe neuron network motifs are the same as those found fn tranacrption and signaling networks This includes FFL and mult-ayer perceptions, “This similarky raises the possiblity that these motifs perform analogous information processing functions in these different networks. “The neuronal network also has motifs nt found in the other networks we have studied ‘One example isthe mult input FFT. structure, which can perform coincidence detection ‘on diferent input ten {i a ofthese cases, networks are a convenient approximation to the complex set of Dilogical interactions. The network representation masks » great deal of the detaed mechanisms at each node and edge. Because ofthis simplified level of description, the ‘etwork representation helps highlight the siriarity inthe cxcult patterns in diferent pars of the network and between different networks. The dynamics ofthe networks at this level of resolution lend themselves to analysis with rather simple models. We care nly that X activates or inhibits ¥, not precisely how it does it onthe biochemical eve, ‘This abstraction helps us to define network motifs as specific functional building bocks “feach ype of network. These bullding blocks offen appet to be joined together in ways that allow understanding of the network dynamics in eems of the dymamies ofeach individual moti Hence, bath an the level of local connectivity pateens ax onthe eveVa CHAP EEIE ‘combinations of patterns into larger cteuts, biological networks appear to display & logics of simplicity. FURILIER READING. re Netw Matis Diverse Netwes Mio tai, S, Kashun, No Levit. Shen-Oet, 8. Ayzenshet 1, Shee, M, and Alan, ‘U-fanny Superamaies of designed td volved networks. Stee, 303° 1588-1502 Milo, fy Shem Ort, 8 tshowts S, Kashlan, Nu Chklowsi, Dad Alon, U. (2002) Network ‘tits sine buldig blocks of eomples networks. Skene, 298 824-827. Developmental Networks Solow, Hand Davidson, EH (2002). Modeling tenseripionl eeustory networks, Hess 2 11S 128 Lawrence, BA. (195), The Making ofa Pye Me the Aken Press . tevine, Mr an! Davidson, £.4 (2005), Gone segulasry networks foe development. Poe. Nat ‘esi Sei 4336-0992. Stathpouloa, Avan Levi, Mt. (2005). Genomic reglatory networks and animal developsnt Dew Call 9479-463, mais of Animal vein. Blackwell Science, Signaling Networks as Conypatational Deviews ‘halla, US, and Tyenga, 1999) Emergent properties of nctwork of blog] signalling path ‘ways, Sionen, 283: 38-37, ‘ay. 1995) Preset moleceles a computational ements in iving cls. Natur, 376: 307-312 ‘Wily, Ib, Shvarsman, SY, and Falenburger, DA, (200), Computational modeling of the TeGteecytoryaten 2 parasigns fr syste iclogy Tens Cel Biol. 13: 49-50 Pereeptrons ane Their Computational Power Heate Ja Krogh A. and Palme, RG (1994) Intradutian to the Theory of Neural Computation. seus Boks Biological Oscillators Baska, N- an eile, S. (2000), Circa clocks limite by noise. Nature, 43: 267-268, Tyson, J), hens Ke a Nowak 3. (2008). Sers, buzzes togles and binkers: dyamis ot exhlatory and wating pathosay i te cell. Curr Opin el fol, 1: 221-23, winiee, A: QUO, Bie Geometry of Biological Tie, Springs Neuronal Netw fC, elegans Hargmann. CA (1998). Newrobiology of the Caenorhabtis elegans genome. Scene, 282 2028-2033, Durbin, RM. (1987) Studies on the development and organzation ofthe nervous syste of ue orhubits lene PhD, thesis, eww mocDaseOR Hope, A Ba. (999), C elegans A Practical Approach is 8. Oxford University Pres EXLRCIS 6.1. Memory in the segulated-fedack network svi transcription fetors Y, and YY, and ¥, swatally fimtion atthe Y, and ¥, promoters i ar OR gate (Vb activate when either X DY, bind the promoter). At time t= 0, X begins 10 be produced fom an intl oncenteatlonof X= 0. Intl, « ¥y~ 0, All production rates re} 2 and dey radation rates area = 1, ll f the activation theeshukls are K = 05. At ime (= 3, production of X stops, ranscription fictor X selves Ir othee the i 8. Plot the dypamies of X,Y, and ¥,. What happs away? Consider the same problem, but now ¥, and Y, eepress each other and X act ates Y, al represses Y, Al time t= 0, X begins to be prodiced, od the initial lovely are X = 0, Y= 0, and ¥_= I. AL tise x = 3, X production stops, Plo the dynamics ef the system. What happens afierX decays away? 40 Y, al Ys afer X days 62. Kinases with double phosphorylation. Kinase Y is phosphorylated by tw input kinases X, and X;, which work with first-order kinetics wit rates vail y,¥nceds to be phospharylated on two sites to be active. ‘he rates of phusphoryiation and dlephosphoryltion ofthe two phosphorylation sitey on Y are the same. Find the input function, the fraction of doubly phosphorylated Y, as. function af the activity of X, and X, Solutio ‘The kinase Y exists in thtee states, with 2ero, one, and two phasphoryations, denoted Yy ¥yand Y, The total amount of ¥ is conserved Yor vey (60 “The rate of change of ¥, is given by an equation that balances the rate of the input kinases and the action ofthe phosphatases, taking into account the fx fe Y¥ and from Y, to Y,.a8 well as dephosphorylation of ¥ 0 Yy AYUMI RY KYM Ow Yow, 63) And the dynamic equation fis Aidt 8 XY AX, Y oY, (52) At steady state, AY, Alt = 0 and Equation P63 yields WX.e 0X) Yay ws)63, 6a, using the weights w= ya and Ww, ya we fin: Yee Valo, Xy #85) (P68) ming esti that a steauy state P6.3 and P6.2 yields d{Y, + YU = % (4, Xy Fy X_) -@ Ys ¥, lv a #5 X) = Yl Ny 90K) esa) Using equation P63, we find Motes tunes 109 Y 52) where eX Xe (es) ‘Thus, the dese input function WY sutra (069) Note thatforn phosphorylation, the Sut function is YJY = whl + a 8) Design a multilayer perceptron with two input nodes, one outpit nade, and as ‘many intermediate nodes as needed, whose output has a region of activation in the shape ofa triangle in the middle ofthe X,X, plane Dynamics of protein kinase cascade, Protein kinases X, Ky. X,act ina signals ing cascade, such that X, phosphorylates X,, which, when phosphorylated, sets ta phosphorylate Ky et, Assume sharp activation funetion. What is the response time ofthe cascade, the lime from activation of to a 505% rise inthe activity oF X,? What isthe effect of the kinase rates on the response time? Of the phosphatase ‘ates? Which havea larger effect on the response time (Hfeinrich et, 2002)? Solon 4 The tate of change of active (phosphorylated) X, is given by the difference hetween the sharp phosphorylation rate By kinase X,.. with ate vy and the dephosphoryla tion process by the phosphatases that work on Xa cate a AKAM ¥,80%.> K-04, ("6.10 65, ‘where 8 the step function that equals one ifthe logh expression X,> Kis tre, and zero otherwise “Thus, X, begins to increase at the time that X,, eroses ils threshold K,, AC this point, X, begins to increase with the familiar exponential convergence to steady state (eg Equation 2.46) X= Oya) -ert-9) (roa) ‘When the concentration of the kinase X; (in its phosphorylated form) crosses the sctivation threshold, i hegins to activate the next kinase in the easeade. Thus, the ‘oniet.of phosphorylation FX, dene eam he fone y saving Ke (yf ll enn] (P62) Yielding + logltt =, Ke (P63) We thus find that a Jog ~ a, Ky (P58) b, According to equation P6.14, the phosphatase rates a, have large effect on the response times, If these rates are very different for each kinase in the cascade, the response time is dominated by the slowest rate, because ithas the largest 1, In conteas to the strong dependence of phosphatase rates, the response time i only weakly affected by the kinase velocities v, because they appear inside the logarithm in equation PS.14 Dynamics of a linear protein Kinase cascade (Heinrich etal, 2002) n the previ- ‘ous problem, we analyaed the dynamics of a cascade with sharp input functions. Now we consider the case of zero-order kinetics. This applies when the activated upstream kinase i foind In. much sinaller concentrations than its unphosphory~ lated target. In zero-order kinetics, the rate of phosphorylation depends only on the upsteam kinase concentration and not on the concentration ofits substrate In is ‘case, we need to analyze a near set of equations: ‘slap eyand the sigaal duration by Joscyava vain {in nus signaling systems the duration of the signaling process is important, in the sense that brief signals can sometimes activate dlleent responses than pra Inoged signals 4, he cascade ist say a plo activity with ample Ay a Jrioaes, Wha the amplitude ofthe final stage in the cascade, A,? 1, Whats the signal duration of X,? «© How do the kinase and phosphatase rates affect the amplitude and duration of the signal? Compare to exercise 64 Solution 4. To find the amplitude et us taken integeal overtime of both sides of lui " 7 Fguation [Note that che integral on the left-hand sk is equal ta X f=) =X (0) Now Because thesis eine att =O and das at grins we av iO) = i) =the Integrals on the right-hand side give rise to amplitudes as defined in isquation ney 8 fy fined in Equat Aan, (P69) (0.20) ‘Therefore, by induction. we find thatthe amplitude isthe product of the kinase rates divided by the product ofthe phospataserate Ba = Wall) Mas =O Vy aly Oy) Ags (roa . 0 Ba Ha Vly 0) A, b. To ind the signal dueation, we take an integral overtime ofthe dynar sion (Bquation 6.15) multiplied by to Gad “mess ‘ (W020) “the let hané-sde integral eam be sved 1 inteyation by pacts to yield “A, (eo.29 Jorn Ihe right-hand side of 26.22 is proportional to the durations of X., and Xp (equation P617) so that we find HAH TMM (v6.2 Hence, we have, dividing both sides by A, and using equation P6.20 to elisninate Boo “ate (6.25) ‘which can be rearranged to yield =a, Hence, the signal duration ofthe final step inthe cascade just the sunt over the reciprocal phosphatase rates ‘é. Werhave jus found that phosphatase rates o,afect both amplitude and duration in veroconder kinetics cascades, ‘The langer the phosphatase rates, the smaller te amplitude and the shorter the duration. In contrast, the kinase rates do not fect duraton tall, an affect the signal amplitude proportionally. This is smi Tar W prublen Ges where we sw that phosphatase ater afc Fiming mich more seongly than kinase velocities In both models, the sum over Va, determines the timing, This principe is wlenical 1 that which we saw in transcription nt ‘works, whose response timies are governed inversely by the degradation/dtution ‘ates These ates are the eigenvalues of the dynamic equations. the strong effect bf phosphatases on signal duration and the weak eect of kinases were demon: ‘rated experimentally (se experiments cited in Hoenberg et al 2005), 6.6, Coineidenes detection. Consider the two-input FFL, motif of Figure 622, the 160 inputs receive brief activation pulses ata slight delay. the pulse of, as duration [Atte ty after the start ofthe pulse, a pulse of, begins and lst for duration 4What isthe minimal S, input pulse duration that ex activate 2 without need for the second pulse of 3? Db. Plot the 2 Abe 1 in which Z shows a response on a phine whose axes are pulse fan dnd interpulse spacing ty 67. Consider the diamond generalization (Figure 67) shat has two inputsX, and X, and 2 single output Z, This two-layer petceptcon pattern has 6 edges. Assume that all neurons are integrate-and-fie’ and each has a threshold K = |. Assure that neu rons have voltage 0, unless the weighted inputs exceed K, in which cae they assume voltage 1. Weights on the edges can be postive or negative eal number. Design weights such that this circuit computes the XOR (exclusve-o) Function, Where Z= 1 ifeither X= 1 ur X,= 1, but Z=0 if both X,= land X,= 1. This function is denoted 7. = X, XOR Xi bb Design weights such that this circuit computes the ‘equals’ Funston, in which 2. ‘only iX, and X, are the same (both O or both 1) and Z:= @etherwise hat is, 2=X, BOX), carrer 7 Robustness of Protein Circuits: The Example of Bacterial Chemotaxis SSS 7:1__ THE ROBUSTNESS PRINCIPLE ee “The computations performed by 2 biologieal circuit depend on the biochemical param eters its components, such a the concentration ofthe proteins that make up the circuit, In living cells, these parameters often vary significantly fom cell to cel due to stochastic cifocts, even ifthe cells are genetically Identical. For example, the expression level of @ protein in genetically identical cells in identical environments can often vary by ens of percents from cll to cell (see Appendix D). Although the genetic programy specifies. 89 1009 copies ofa given protein per cell in a given condition, one cell may have 800 and its neighbor 1200. How can biological systems Function despite these variations? Tn this chapter, we will intoduce an important design principle of biological circuitry biological circuits have robust designs such that their essontal function is nearly indepen dent of biochemical parameters that tea fo vary from cll to cll ‘We will eal this priteiple robustness for short, though one most always state what prop: ertyis bast and with respect to which parameters, Properties that sre net robust recalled fine-tuned these properties change significantly when biochemical parameters are varie Robustness to parameter variations is never absolute: it is a rcative measure, Some mechanisms can, however be much more robust than others Rebustness was suggested tobe an important design principle by M, Savageau in theo retical analysis of gene circuits (Savageau, 1971, 1976) H. Kacser and colleagues experi: ‘mentally demonstrated the robustness of metabolic Dhxes with respect to Variations of enzyme levels in yeast (Kaeser and Burns, 1973). Rohustness was also studied in a dif ferein context the patterning of tissues as an egg develops into an animal, Waddington 1sHGR 1 tte sta, ace sepetn cv pete wih aetna a ay Gm studied the sensitivity of developmental paterning to various pestarbations (Wading "om, 1958). fn these stidiey, robustness ss called cavalization and was eomsered a the level ofthe phenotype (the shape ofthe organism) but nt at the lve of biochemical mechanism (hich was largely uke a the tine), Recent work has demonstated how bropery designed bioehesnial citculey can give rise to robust and precise patterning, “This sujet wil bo discussed inthe next chaptee Feere we will demoustrate the design principle of robusiness by using a well-character ‘ed protein signaling network, the protein excut that controls bacterial chemotaxis. We will begin by describing the bislngy of bacterial cheniotaxs, Iisa relatively simple peo- totype for signal transduction circuitey in other eel types. Then we will describe models and experiments that demonstrate how the computetion performed by this protein circuit 4 male robust 1 changes in biochemical parameters, We will sce that Ue principle of ebastness cat help ust rule outa large family of plsible mechanisms and to hom ‘on the corret design 7.2 _ BACTERIAL, CHEMOTAX! 2.1 Chemotanis tehavior ‘Whe a pipette containing nutrients is placed in a plate of swimming Escherichia col bac> {eri the bscteria ate arated to the mouth ofthe pipete and form a cloud (Figure 71). ‘Whew pipette with noxious chemicals is placed in the dish, the bacteria swim away fro the pipette. Ihis process, in which bacteria sense axel move along gradients of specific chesnicals. i called bacterial chemotants ‘Chemicals hat attract bacteria are alle attractants. Chemicals that deve the bicteria ‘vay are called repellents, cl van sense a variety of atractans such as sug and the ‘sino acs serine an aspattate, and repellents, els metal fone and the arsine ack leucine, Miow bacteria species show chemotaxis, andl soine can sense and move toward stimali such a ight (phototaxis) and even maghcte fields (magnetotaxis), Bacterial chemotaxis achieves remarkable pecformance considering the physical ini tations faced by Uke bacteria, Bacteria can detect concentration gradiats an small as a change of one molecule per cll volume per micron and function in background con satrations spanning ove five odes of magnitude. All this is done while being bulleted by Beowrnian notse such that if he cell resco swim steuight for 1D seg, ts erentation #3 randomized by 90° on average How does F. ca manage to mowe up gradients af challenges? 1 is evidently t00 YOR HOW BACTERIA THINK _ tractants despite these physicat to sense the gratent along the length af its own i SS mS COT 7. Taormina ht sons anes ring $e win ws Suk nvtonnent Rens are pets of ony tengo, ble ae ei eves i et ‘vation randomized Dong chemaaes, beets ede te tng egy wh sing coisofasrcrans body! the answer was discovered by Howard Berg inthe carly 1970s: E.coli uses tempo ‘al gradients fo guide is motion. 1 uses a based-random walk strategy to sample space and convert spatial gradients to temporal ones. In liquid environments E cléswims in 8 pattern that resembles «random walk. The motion is composed of runs, ia which the cell keeps a rather constant direction, and tumbles, in which the bactriom stops and randomly changes direction (Figure 7.2). "The cuns last about L sec on average and the tumbles about 0.1 sc, ‘To sense gradients, F coli compares the current attractant concentation 10 te eon- centration in the past, When £, coli moves up a gradient af attractant, it detects a net positive change in atretant conceatration. As a result, it reduces the probability of a tumble (it reduces its tumbling frequency) and tends to continue going up the gradient ‘The reverse is true for repellents if detects that the concentration of repellent increases ith time, the cell increases its tumbling frequency, and thus tends to change direction ‘and avoid swimming toward repellents. Thus, chemotaxis senses the temporal drivalive ofthe concentration of attractants and repellents. ‘The runs and tumbles are generated by dffeent states of the motors that rotate the bacterial ages. Each cell has several Nagella motors (Figure 73: se also Section 5.5) that can eotate either clockwise (CW) or counterclockwise (CCW), When te motors turn ‘CCW, the Magee rotate together in a bundle and push the cell forward. When one of ‘the motors turns CW its flagellum beeaks from the bundle and causes the cell to tune About and randomize tsorientation. When the motor turns CCW, the bundle is reformed and the cll swims in arew direction (Figure 74). 72.2 Response snd Feact Adaptation “The basic features of the chemotaxis response ca be described by a simple experiment. In this experiment, bacteria are observed under a microscope swimming in aliquid with SESE pee etn yt tan inc tne ncaa hl Ths ‘steht et te Ln abt oom ese poder alana a las fre fo cle rls Teh astro he hcg ge ~ 3 mk ih ain le pl ny ng i ange cba cy wa ce tome ‘defn and bbe pees onthe apa empl rats eesf 'S, we meee Ley Men SED nici Tid RALOOR ee Ger ase HHGURE 73 The bacteria gea moto Right paca eston mise omc ge ofthe i ‘els nr. Com a 2009) no gradients, The cells display runs and tumbles, with an average steadypstate tumbling frequency fon the onder of f~ 1 se ‘We now add an attractant such as aspartate to the liquid, uniformly in space. The attractant concentration thus increases at once fram zero to but no spatial graents are formed, The cells sense an increase in attractant levels, no mate which direction they are winning, They think that things are getting better and suppress tumbles: the tumbling frequency of the cals plumnmets within about 0.1 sec (Figuce 73), Aftera while, however, the cell realize they have been footed. The tuskling fiequency (of the cells begins to increase, even though attractant is still presen (Higare 73). This process, called adaptation, is commen (o many biological sensory systems. For example, when we move fom light to dark, our eyes at fest cannot see well, but they soon adapt sense small changes in contrast. Adaptation in bacterial chemotaxis takes several seconds to several minutes, depending onthe size of te attractant step" ‘acterial chemotaxis shows exaet adaptation: the tumbling frequency inthe presence bfattactant returns tothe same level as before sttractant was added. In ether words, she steady-state tumbling frequency is dependent of attractant levels, ig egy vats acl fer any ncn sl (rt 98 ewan a Hc ota tele S\, iti So \ <3 GUE 74 Bacteria rata ues aereae othe eto dsctin othe als ters When al motors spn coumercackwise (CCW) the apelin band an the ls eee ora Wh neo more mors te lacks (CH), the el umes an ome st rettion, Te sete symamics of sng moar rom CCW to CW and bck ca be sec hy tethering acel to sone by on "gel hoot, shat te mrp the ete el aly euelesa erips few Mer ee 1 loge vicousdragof the dy). HGR 75 Avragetombling reper os paplaion a el expsed ane t= Sto ap wl of Sonraing strc (aspartate). Aer? eaten wry pase a conta concent ‘on Adepution eas hat thee of he tin i geal frgren despite conned presence vacation ea ert eter pesmi evel, ht asta tate ambling aun ht dos not depend om hele of rca If more attractant is now added, the cells again show a decrease in tumbling fre |qency, followed by exact adaptation. Changes in attractant concentration cat be sensed slong as altractant levels do not saturate the receptors that detect the attractant. Foract adaptation poses the sensory systern at an activity level where it can respond ‘to multiple steps of the s ‘other atractants and repellents that can occtir tthe same time. It prevents the system we attractant, as well as to changes in the concentration ofTAU CHEATER 7 (auras . . bot ay f° (Ke A [Oo nba eq) GUE 76 the chemotaxis al transdustin network efrnatin aba the che evonsie i voce te the elh by reepre nul ath aeprtae veo Ta which spn he mera Te ‘hemereeptr ran npn the cl with the KnstsCaeA (AP athe dap peta Cal? (WN Chen psporybies se? nd Ue aelerspespory (grou to Chek (Y) «die wer Senge tla. The phosphorylated fn of Che trace with the ela torso duce ble Te ‘at of Ch deposphoryiton i wrest enbancee by eZ (2. nding of stractans to theres ireses hee of Che phosphoryl abd arbing seduced. Adapt epee By cages Inthe evel metaton ef the cemereceplacsmetiyaon icra the ate of Ce phosphoryl, ‘pal of enyncs, Chel i) 0 Ce (a an eves) ups. To lp oa atta, toslition ofthe recetrs nt ie oven he mppreston of satya bythe at tac ining Ce enhances the demining scot Che by pompboating Cet. (nom Ake ais) ng away soma favorable steny stale tumbling frequency thot is required 10 eslciently seam space by random walk 7A THE CHEMOTAXIS PROTTIN CIRCUIT OF © COL ‘We now look inside the # coli cell and describe the proteis clecuit thot performs the response and adaplaton computations. The inp to this cireuit isthe altractant concen- teat, and is output isthe probably that motors turn CW, which determines the cals tumbling frequency (Figure 76). The chemotaxis circuit was worked out using genetics, physiology, and biochemisey starting with J. Adler inthe late 1960s, followed by seve Tabs, including those of D. Koshland, S. Parkinson, M. Simon, ). Stock, aod others. The broad biochemical mechanisms of this circuit are shared with signing pathways in all types of cls Axtractant andl reellent molecwles ae sense! by speciatined detector proteins called receptors. Each recelor protein passes throu the eels ianer aerate, ad as one part outside of the cll siembrane and one part inside the cell. Ir can shus pas in. ration fio the outside tothe inside ofthe cll. the attractant and repellent molecules sind by 3 receptor ae called ts Wigands. -cols hs ive typos oreceptns, each of whic can sense several Higa, there are & tots of several thowsand receptor proteins in each cell. They see localized ina chaser {he inner mena, sch that Tgand nding to ane reeentor appears to somehow afect the sate of neighboring receptors, Thus, 3 single Higa binding event is ample, because ican affect more than one receptor (ray, 2002), increasing the sensitivity of his molec lar etection device (Segall eal, 1986; Jasujae al, 1999: Soulard Berg, 2004), Inside the cll each receptar is bound toa peoten Kinase called CheA.! We will cone sider the receptor ant the kinase aa single entity alled X. X transis rapidly Between toc sales, active (lenotedX°) and inactive, on stones of microseconds, Wher X is active, Xi causes a miodifcation toa response regultor protein, CheY, which diffuses ‘nthe cll. "This modiication i the addition ofa phosphoryl group (PO) to Che¥ to form phospho-CheY (enoted Che¥ P). his type of mexiiation, called phosphorylation, is sed by most cypes of ells to pass bits of information among sigasling proteins, as we saw iy Chapter 6, Che¥-P can bind the flagella mator and inceease the probability that sites from CCW to CW rotation. Thus the higher the concentration af Che¥-P, the higher the tambling frequency (Clszel tal, 2000, ‘the phosphorylation of CheY-P is removed by a specialized enzyme called Chez. At steady-state, the oppesing actions of X* and Che? lead toa steal state Che¥-P level and a steady-state tumbling frequency: hus, the main pathway in the eieuit is phosphorylation of Che¥ by X* leading to tumbles. We now tumn to the mechanisms by which attractant and eepellent ligands ean aifect the tumbling fequeney. PB. Ateactanis liner the Actvily of X ‘Wher ligand hinds receptor X, it changes the probity! thot X wil assume is etive Sate X* The concestrabon of X ia its active state is called the activity of X. Binding of| an attractant lowers the activity of X. Therefore, atlsctants reduce the rate at which Phosphvyslaea Chic snl vel of GeV drop. Av woul le probability of CW saute ‘otation drops In this way, the attractant stimulus results in reduced tumbling frequency, 0 that che cllsReepon swimuming inthe eight direction, Repellents have the eewers effect they increase the ativity of X, resulting in ineeeased tumbling fequerey so that he ell swinis away from the ellen. These responses occur oes apse ar am wt the rer pee spyng hat maa ese hc an scr aes opt. ae ein aed ode ee abt heen rants Deen 4X ag foe 3 encod nee Tere any ne mtn sgegans air cee ey X nce yea net ay Manan (sks Homi 8 Ste ib, Kye 3 208), Probyte gaia {rican is nes tyes e he tym tinewithin less tham 0. see, The response time is mainly limite by the time i takes Che¥-P to diffose over the length ofthe cells, fom the patch of receptors at the cll pole where Che is phosphorylated to the motors that ae dsteibuted all around the cll, the parbway from X co CheY tu the mator explains the intial response m figure 75, which attractant leads to redaction i tenting, What causes adaptation? 2.82 Adyplatinn bs Due to Slow Motiication of X Tha lvereases Is Activity “The chemotaxis circuit has a second pathway devoted Lo adaptation. As we sav, when attractant ligand binds X, the activity of X is reduced. However each rector has several biochemical “buttons” that can be presse to increase is activity and compensate forthe cefect ofthe attractant, These buttons are methylation modifications, in which a methyl sirup (CH) ie added to for ur five locations on the receptor. Vach rept ‘between zero and five methy! modifications. The more metal groups thal are added, the bigher the uetivty of the receptor. ‘Methylation ofthe receptors is eatalyzed by an enzyme called Chek and is removed by an enzyme called CheB. Methyl geoups are continually added and reinoved by these two. ‘antagonistic enzymes, regardiess of whether the bacterium senses any ligands This seem {ingly wasteful eye hasan important funetion: it allows cells 0 adapt ‘Adaptation is carried out by a negative feedback loop through Chel, Active X acts to phosphorylate CheB, making it more active, Thus, reduced X activity means that CheB is less active, causing a reduction in the rate at which methyl groups ate removes by Chel ‘Methyl groups are stil added, though, by Cheat an unchanged rate, Therefore, the co centration of methylated receptor, Xu. increases. Since X, is more active than X, the tum bling frequency increases. Thus, the eceptors X frst become ess active doe i attractant binding and then methylation level gradually increases, restoring X actviy ‘Methylation reactions are much slower than the reactions in the main pathway from X to Che¥ to the motor (the former are on the timescale of seconds to minutes, and the latter on asuidsecond timescal), The pzotein Chel is presenta low amounts in the ce shout 100 copies, and appears 1 act at saturation (zezo-orderkinetis), the slow rate of ‘he methylation reactions exphins why the recovery phase of the tuntling frequency ‘during adaptation is mc slower than the initial respons, The feedback eieuit is designed s0 that exact adaptation is achieve. That is the increased methylation of X precisely balances the seduction in activity caused by the atractant, How is this precise Dalance achieved? Understanding exact adaptation is be jgoal of the models chat we will ext describe i have 74 TWO MODELS CAN EXPLAIN EXACT “ADAPTATION: ROBUST AND FINE-TUNED (One can develop mathematical models to describe the known biochemical reactions i the chemotaxis cireut. We will nuw describe two different models bast on this bio chemistry. ‘Ihese are toy models, which neglect many details, and wase goat is 12 ‘understand the essential featuses a the system, Boh movlels reproduce the asic response lof the chemotaxis system ply exaet adaptation tn one model, ena adapta Fine-tried and depends om a precise balance of ailferent biochemical parameters. fn the second madl, exact adaptation is robust and aceurs for 3 wide range of parameters, FAA Fine-tune! Mule (Our frst model is the most direct description of the biochemical interactlons described above. In other words. i isa natural first model. ladeed,chis model i a simplified fox ‘of theoretical model of chemotaxis first proposed by Albert Goldbete, Lee Segel, and collegues (Kinox tal, 1986), his study formed an important bass for Laer theoretica) ‘work on the chemotaxis system. In the model (igure77), he receptar complex X cas bocome methylated X, under the action of CheR, and demettylated by CheB. For simplicity, we ignore the prcise number ‘of miei groups per receptor and group together all methylated receptors into ne va able X,. Only the methylated receptors ae active, with ativily a per methylated recep- tor. whereas the unmethylated eeceptors ate inactive. ‘To describe the dynamics of receptor methylation, one needs to model the ations of the ‘thylating enzyme Che and the demethylating enzyme Cheb. The enzyme CheR works at saturation, (What is ata rate that is independent ofthe concentration of is subsrate, ‘with rate Vp. In contrast, CheB works with Michaelis-Menten kineties (readers not faril- iar with Michaelis-Menten kinetics wil find an explanation in Appendix A). Hence the rate of change of Xi the difference ofthe methylation and demethylation eat: oar aR VaBX AK 4X.) an, ‘The parameters R and I! denote the concentzations of CheR and CheB, At steady state, AX fd = 0, the dynarsics reach a steady-state level of methylated receptor: "GUE 7:7 rine tuned nahn for eat opto. Reteplrs te methylated by Che od det ‘ety Ch. Meth ect teu wit atm) eae the phorylation of Che edie 0 fumbles. When atacand the ati of each yn een edo and abi fede Twat, te etry Hf Chl a recede ote wee fk oy te sy Th, Re eo ‘Staton of mye reeers aly ieee i The sing equeey fla to he pest "ste pain ents ting Retest odction a Chel acti ant he edi i "riya ct uate Ning. e ty eran e pesaeeKV RIVE VR) 42) Recall that the unimetilated receptor bas zero activity, whereas receptor, revlting ina total steady state activity of bas activity, per A Xe steady-state activity with wo attractant (74.3) ‘The activity ofthe receptors Ay deveribes the eate a which Che¥ is phosphorylated to ‘create Che. The phosphorylated messenger Che¥-0, im tea, hinds the mator to gener ate tumbles. The activity Ay therefore determines the steady-state tumbling frequency, FAQ. Nove imagin bind attractant iat saturating attcctant i add tothe cals, so that all ofthe receptors igand, Th attractant causes receptors to assime their inactive conforma tion. As result, the ativity per methylated reeeptor drape to a, ay Therefore, the total sctviy al short times afer ateactant is added drops to a low valu aXe aay thus, the total ativity is reduced aller addition of attractant, A, GUE 7 the Haast mechani fr xt adaption, Unmet eceytomare ethyately {Chet at cnsant te Detain di to Cl wha ony teste melt espe Mey ited cepts athe with} ral bet ative inact eth ome are sei an Atacand increas the ebb obese ate whens ees erase he Prot to become eve The see eer etaloe he phnpoeylation Che lang ames Wien atasent sade niny ative ces rai bet inet alesse ee reich sce nombre epee ot Che as ste work ol he the detonate drops. nce Che cominnes to mort cnstnt ate he tel uber fm sed sectors ntetes Ths Incase onl ss when deme cae val lps eto fa, tat hen the amber of ate rcs run of pestiny e,ts. eae aan secre Bie the onceataion of ase esr ts il ht he demetytonrate Thecantnt ntl “The model ie hased on two key features, First, Che must work a saturation, Second, ‘CheB can only demethylate the active recepiors, Xy* (Figure 78). To repeat, Che does rat work on the inactive methylated receptors, This lend tothe following equation for the total concentcation of methylated receptors (both active and inactive ea) ‘where R and 8 denote the concentrations of CheR and CheB, Note that the Michaelis Menten term for CheB contains the concentration of its substrate, active receptors Xy' “The steady-state uf this dynamic equation occurs when d(X,,+ X,)Alt = 0. At steady state, the value of X,* reaches a point wheze demethylation exaetly balances the canstant fox of methylation (743) (74.16) ‘When attrctant is added, it binds the receptors and decreases the probability of the sctive state. Therefore, the number of active receptors A» X,* rapidly decreases. This ‘causes the abrupt initial drop in tumbling frequency that is ebserved inthe experiments (gure 710) ‘Afler this sharp initial response, adaptation occurs due to the fact that Che only works on the active receptors, The rate of demethylation by Chel Ts reduced beeause of the decrease jn Xy eaused by the attractant. CheR, on the other hand, continues to meth late receptors at constant rate, Therefore, the total mumber of methylated reeeptors gradually increases ss at ol et » saan < fn o ® 21) Activity nario he ot mela esponseo ation of saturating strata at ise (5) Met parameter = lV and Vy 3: arnt wh ede by tn 42 Tantaapatio preserved Noe a the uc othe tend tte taming Sequecy 8 Bete ‘nd dependent mol paralaay nied saps Kes ih ae etn tet mate resp ‘nts seid wn teckel neste cra Chek Snicket nase Ay VB-VAR oats This activity is equal tothe pre-attractant acti not depend His eves y. Thus, the steady-state activity dacs A cary ow does this mechanism wort the eri meats ate is fx of methyation ‘eto Chel set ayant countevlox of demelhpation by Che that directly epends on theactivity A= Xe AL steady stat, the sumer of active receptors always lusts isl 39 that demetiation balances the eference flux of methylation In other words the active spi "etn the ed put of Last 77 0 ae wht the ate lve The aclvity AX," reaches steady-state abe that doesnot depend onthe liga s, Exact adaptation i achieved. Figure 7.10 show the djnamic ofthis model for ‘vo sets parameters, in which Chet levels ate varied by ator of seen that the steady-state activity changes, but adaptation remains exact. . [Exact adaptation occurs fora wide range of variations in any oF the parameters ofthe cela ipl depends on thx pants tnaher words tea «a fine-tuned (vature of this model (Figure 7.10), Exact aday which the say sate 70) Fxat adaption, in whch hse ae doesnot depend on ligand lew irs robust feature of the me cn the precise values of the biochemical parameters. me fos there ae nits to rbuatoas or eam, wn Vl excels Vy he saturation ssn envy help nds on Rausing eat apalion Jn sod ns od dps oma sump that Che venice soi a nh tive stat Ths spec shea lta Wee ob edge. The ‘Simp tht Chet wos ny sac repos tetas en caps can be exquisitely specific in dlcriminating betwen moleculat sates, Relaxing this ‘Ssunptonyllow!gasal ti tee fr Chl action on ince cele «loss of exact adaptation by a factor on the order ofc poses 5 Hobust Adaptation and intl Feedback. tend enn tp dain est othe acy, ahr than tse ute yc nthe ee of Che The eg Geedlbuck loop therefore acts directly on the variable Ww be controlled. “ue she iret feck inthe edbust mechanisn of exact adaptation fs elated to te ei cring conte principle of antegralFeiback (i tal, 2000. Ln ings eon’ 3 eects envelled hy a sgeal that integrates over tine the errr betsees the expat sea desired utp his type of feedback is guranteed to gue the Levice to the see ut level, eres variations i the spe parameters, becnnse lire the negral ofthe evcrgrows witout bound. Moreoes, it any cases etegra feedback, an he shown to be the only robes solution o this peublem. the integrator i bacterial spermavants Lat effctvly sums the erro iv activity (the activity minus the steady state sEhty is the methylation fv of the receptors, The properties of ince esc Ghenutanis are examined in exercises 72 al 73. JAA Typwedinents Show Sh Fssect Adaptation Is Robs), Wheres Stel Sate Ac ivly and Adaptation Tanws Are Fine-Tunes “Anexperintal ett robustness erployed genetically engineer Eco stains which ‘Aioweel controlled changes inthe soncenstation of each ofthe chemotaxis proteins (Alon veal, 1999) This control was achieved by fst deleting the gene for one chemotaxis pro: tein ior example, Chel Irom the eomosonne, al ther introvcing into the cll a copy tthe gone under cone ofa inducible prometer he far promoter). Thus, expression of the protein wus contrlld by means of an exteraly added cemial induce (TPT). The trove inducer that vas added, the higher the Ce concentration in the cells. In this ways Chef levla wore varied from about 0.510 50 times thir wild-type levels. The popstation respons of these ellsto a saturating step of attractant was mocitored wsing veo micros Copy om svimming dls, The experiment was carried out with changes in the expression evel of diferent chemotaxis proteins, te was found that ‘he steady-state tembling Frequency and the adaptation time var sed with the fevels ofthe proteins that make up the cheaotaxis network (Figure 7.10). For example, steadystate tumbling frequency increased wilh inereasing Chel levels, rhereas adaptation ime decreased Despite these variations, exact adaptation remained bust to within experimental erro. These results support the robust model for exact adaptation, INDIVIDUALLY AND ROBUSTNESS IN BACTERIAL CTH MOL Spudich and Korklond (176) observed. that genetical dential cells appeat to have a taividua chatactee as they perform chemotaxis. Some cells are “nervous” and tumble more frequently than others, eherea other cells are “relaxed” and swim with fewer tum- ‘les than the norm, These individual characteristics ofeach cll ast fr ens of inate “The adaptation tve to an alteactant stints also varies from cello cl, Interestingly, these two features ve correlated: the steady-state tumbling frequency Fin « given calli inversely correlated with iis adaptation ime, thats. £~ I “She robust mode’ fae bacterial chemotaxis ean supply an explanation forthe varying hensotaxe personalities of E, col cells, This is basel on the eall-cll variation in chemo~ taxis protein levels, and particulany in tbe last abundant protein in the system, Chek. Vavations in Chelt Meet the cusbling Frequency Fand the adaptation time x48 oppositeane bos 2 op 8 Aetpaon dn fin) Sedyantng gemnn engese CreRidesasion % {GUI 231 apres ef tbs in el cena. The poi Che was epee silt eee ms coiled exresion mip nde promo abd the mene eng eqn ofl population nas mead wing ko meepy aleen ee at Co peti a the wile bcs apn prcon te a oat hea wt nd ater trang rota mM apie) Asp ine sth tne aseu Pe seul sate tong rie afer etrling arco om Adan ne saree bin eiccy vid wih Ch, wher adpton renal ac Wipe nbs eres a thse abort a ttn) om lta eB) ‘ieections (Figure 719). The Barkat-Leibler model with muhiple methylation sites suge bests that f~ Chelt and x ~ U/CheR. ‘hus, the model predicts that {~ 1/. explaining the ‘observed correlation in these two features (se slved exercise 7. Despite the cell-cell variability in tumbling frequency, the vast majority ofthe cells in “population perform chemotaxis and climb gradients of attractants. Om the other hand, utant cells that have ssl-type tumbling frequency but eannot alapt precisely (uch 8 certain mutants in both Chel ad Cheb) are severely defective in chernotaxs abit [Sse tion fhe mete perl P yagi Shima ta ity, Evidently, tumbling fequeney nes not be precisely ted for successful chemotaxis, ners exact adaptation is important for most ligands. In summary, i appears that the bacterial chemotaxis circuit has a design sch that a key feature (exc aaptation) is robust with respect to variations in protein levels. Other fentures, such us steady-state activity and adaptation times, are fine-tuned, These later rinsiccell-call variations in peo: Jeatures show variations within a population due to ‘cin levels. Because ofthe robust Ucsigo, the intriesic variability inthe cells protein eves doesnot abolish exact adaptation. ‘Ass theorist one can usualy wete many diferent models to describe a given bisogi cal sytecs, capecially if sone of the biochemical interactions are not Fly chaste ic Of these models, only very few wil typically be eabust with respect 10 varinions in the components. Thus, the eobustness principle can help narrow down the range of models ‘that work un paper to the few that can work i the cll Robust design isa important fa tor in determining the specific types of circuits that appear in cells In the next chapter, ‘we will suady how robustness constraints can shape the circuits that guide patter form: tion in embryonic development FURTHER RFADING 7 ‘loa, U, Suets, MC, Backal, Nand Leible, 8. (1999), Robustness in bacterial chemotaxis Nature, 397: 168-171 Barkal. N. end Leiber, S, (1997). Robusiness in simple biochemical networks. Nature, 387 Berg, H.C (2003 Fol Motion. Speinges. erg, 16. and Brown, D.A (1972), Chemutaxsin Eschercla cl analyzed by thre dmensol teacking, Nature, 25% 500-504 ‘eng snd Parcel, M1977), Dhyses of chemoreceton, Hops. 20: 193-219, Kitano, (200), ogi] bso, Nat Rev inet $: 826-892, Knox, BE, Devevtes, PN. Goldbete, As and Sept A (1986) A evleewar mechanism fr sensory adaptation hase on ligand indice! receptor modiicalion, Proc, Natl. Ac. Ss USA. 83:2345-249, Kollman, M, Lavdok, L, Bantholome,K, Timme, Land Sour, (2008), Desig priacpes cof bacterial signalling network, Nature, 43: 504-507 Spudich, 1, and Koshland, DE, fe (1976). Non-geneti indiiduaity: chance i the single el Nature, 262: 467-8. -YIAEMC Hung, ¥ Sito, MEL, and Doyle, (2000). Robust perfect adaptation in boctera che ‘ais thegh intgral feck cont, Prac. Nal Aen, Sh U.S.A. 97 4649-468, cal ting eqs i wan umn the aaa sen igh ome ‘eran 93) Ate conarton eneEXERCISES 2 Robust mented wit two wethylation vies, The receptor X can be methylated on two Positions, and can thus have zero, one, or tw anethy groups, denoted X,, Xj, and Xp the enzyine R works al saturation (zero-order kisetis) ts methylate X_ and Xp. 'The demethyating enzyme B works only on the active reegpor confurasation, removing metiyl groups with equal eae from X* and X,*. For simply, assue Ua B works wit st nrder kinetics, The eesctions are: methylation XX, at rate R Vy XUN # Ky the fast factor occurs because R is distributed between its substrates X, and X, methylation Xy-o X; at ate R Vy XO, + XD X, 2X" rapid tsi ata rate that depends on the ligand level Xy=2 Xs eapid transitions ata hat depends on the igaal level de-methylation OX, strate BY, X desmethyation Xe OX, arte BY, |. What the steady state activity A = X," + X,*? Dacs it depend on the concent Udon of ligand Is there exact adaptation? Estimate the adaptation time, the time needed for $0% alaptation after adlion of saturating attractant. Note that to adapt to saturating attractat, vetutly ell of the receptors ned to he doubly metal © Spudich and Keshland (1976) foun chat different cll in a population have dif ‘erent steady-state etivitcs an different adapation times. Moreover, tes LW features were found to be correlated: the higher the activity A, the shorter the adaptation time t ina given cell, with A ~ Ls, Explain this finding using the ‘model, based on ccll-cel variations in the concentration of R (Barkai and Leiber. 1997), Solution: 8. The rates of change of the doubly methylated receptor concentration and the ‘ponnetilated receptor concentration ae Axe Xie ViXVK + XJ= 18 Va Xt en EXAM RVG XI, NDB VX wr) Swbtracting these two equations yekle dO Xt “AX fa Va BY aC EXPE RV. -BY,A (PRB) Ihe activity A= Xj + X,* is Unerefore (etg Ul terms to 2000) AGE RV YB, (ra) This aetvity does not depend on the ligand concentration, "Userefore, this anism disphysexset adaptation wh In the case of saturating ligand, all receptors inal f thee form bind attractant, ligand. the attractant veduces the activity of all methylated receptors, and thus at intial times X,* is small. lo addition, when adaption is completed, X/ is small because the majority of recepors need to be doubly methylated in order torhalonce te stcong inibitory elec of the saturating attractant, Thus, it semis that X,* is reatively small throughout most of the dynamics. Sie Xi small tion Mux rom X,*10 X, small. ence, o.a good approximation, X, dynamic ellect only a reduction de to the action of Che, because the teem with B in Equaion P22 is neigible: the deme OX AL RV XIOG + XI 75) so that X, drops with time, At inital times (before attractant addition), let us denote by athe frsction of X, among the possible substrates of CheR,q = XIX, +X) Thus, the inital slope ofthe drop in X, is = q R Va. The adaptation time to saturating ligand (ime to recover to 50% activity) i the time needed to itll ‘enough methylated receptors to restore activity, atthe expense of most of the unmethylated ones, The cf appeosimntely the sme for X, to decline 0 50% ‘of its intial value. Tis adaptation time is equal 10 the numberof mehylation swsctions needed (ht fe, mitalations equal ts 50% of%,) divided bythe ae st ‘which they occu, namely (ignoring the changes in over this time) +05 KG RVy co) “Thus, the adaptation tise becomes shorter the more R enzymes exist inthe ell. “This makes sense Because ‘occurs and the faster the adaptation, Note that the single methylation model discussed in the text has a different adaptation tine, governed by Band not R, Us because we cannot ignoee the e more R enzymes there are, the faster methylationMx rom to Xy- whieh is necessary to proce exaet ala ‘methylation model. But B governs the adaptation time only if we restrict our selves to single methylation site as we did for laity in the text In realty there are multiple methylation sites. ‘The adaptation time is generaly governed by R {in models with more than one methylation site (Barkai end Leiber, 1997). 1a experiments the adaptation time i found to decrease with R (Figure 712), in agreement with the multisite moves lon the single {We saw above thatthe adaptation time varies as + 1/8 (Eaton P76) and the sendy-state activity varies as Ay ~ R (Equation P24), ‘Thos, if Ris the protein Wit the largest variation between genetically identical calls, one would expect Uhat Ay ~ 1/5, a8 observed. “Ihe protein Ris the least abundant chemotaxis sig- naling protein in ent, with on the ord of 100 copits per eel, whereas there are on the order of several thousand copies of Chel, Che¥, Chet, and Chea per cel: Che may therefore be the most prone to stochastic varatens 712, Integra feedback. A beater heats 2 room. The room temperate T increases in pro Portion tothe power of the heater, Po other sources of heat, S, ard decreases cic to thermal diffusion t the outside a a rate proportions tT Tak =aP +80 7) ‘An integral feedack device is placed in onder to keep the oom tzmperature a & desired point T, In this feedback loop, the power to the heaters proportional to the integral overtime ofthe error in ermperatare, T=, Pao KUT. at P78) This fedback loop thus reduces the power tothe heater if the root temperature is {oo high, T > 7, and increases the power when the room tenspersture is too lo. "aking the time derivative ofthe power, we in APAle -K T= 1 (79) 14 Show that the steady-state temperature iT, and that this sead-state does not {epend on any ofthe system parameters, incleting the rooms thermal coupling to the eater, Une addtional heat sources, S, the room's thermal coupling with the outside, b, othe strength ofthe feedback, K, In other words, integral fed: back shows robust exact adaplation af the room temperature, 1b, Demonstrate that integral feedback is the ony solution that shows robust exact adaptation of the room temperatucs, out ofall possible linear contol systems “That i assume a general linea form forthe contraler: 710) JTree and show tht teat edback asa tc esteem ey induce br eben rx oot Ing feck chem Drosha npn Fra hemottnconans nea etiace What the neg inh bo seals (ta, 2007 sain an tnatner model Che works uration ad Che works i r2eitopen hehe The eo haf lmber eh Seedacen Bence ss epwenby he erence bese he Det denon tes ax cnn) VyR-VaBA “This can be eewritten in terme of the difference between the activity A and Its sendy stat value Ay A yldt= Va BAAS) ena where the steady-state activity is y= VaRIVaB nas) “Te toa numberof methylated receptor Xan tus ot asthe inept in the system that integrates the errr in activity overtime (in analogy te Equation P78) Xap BJA Adal wna the sty & is analogous othe oom empertre in pole 72. Tb complet he analogy with problem 72, ws weit a deta eguation fr the ete of change of activ, A= X57 The numberof methylated active receptors Xe sneeaes dic to transitions font X10 Xpt ata ligad-dependent rate, KD, he number Xa" Uecreases due to the Gemethjlating action of CheB and due to transitions to inactive ate X, a a gand-dependent rate KD. The dymamis of Ny! = A ave therefore given by the sum aver the eates of al ofthese transitions with appropriate AMIE = KD X= KW) A= Vy BA (ri)7A, We want to eearrange this equation 9 thatthe fist term is proportional 19 Kye Xq.4 A (analogous fo the heater power I in exeteise 72s Equation P77), For this porpose, we add and subtract KDA to Bind JAK KD XRD A= Wy ha (eng) ‘hws, we end up with an integral feedback syste P79, in whieh | yous to Equations P27 as AANA X= bA en) AX ull KOA A,) was) wheres =K{),=K'C) 1 K()-+ Vy Band K = VB “To restate the analogy, think of A as the temperature and X,, a the power to the hheater in problem 7.2. As shosen in problem 72, the steady-state activity A, does not depend on any of the parameters, b, or K, and in particular on the ligand level that enters only through (1) and kin the parameters aand b, Thus, Ay dos not depend on the level of attractant (or repellent), and exact aduplation is acheved Zers-onder ultrasensitvty (Goldbcter and Koshland, 1980); In this exercise, we will ‘see how two antagonistic enzymes can generate a sharp switch, A protein X car be ina modified X, or unmodified X state. Modification i carried ut by enzyme E,, and de-miodifcation by enzyme E, The rate V, of E, is constant, whereas the rate V, (of E 5 governed by an external signal, Consider V, as the input and X, a8 the out. ppt ofthis system, (@)_ Assume that E, and F, work with fistorder kinetics, What isthe output X, a Function of input (8) What is the sonstivity of this relative change iV. i, defined asthe relative change iv X, per vox, s0x,.vp- tet (©) Assume now that and E, work with zero-order kinetics, What is X, 98 fun: tian of V)? Note that X, + X, cannot exceed the total concentation X,y 2) What isthe sensitivity ofthe zero-order circuit? txplain why tis called “zero- order ultra-sensiivity” (© Compace the switching time (de to 509% change in X, upon a change it ¥\) between the cases of fd) and (e) above 78. oust model witha single methylation site Consider the model of Section 74.2 he fpethylated receplor tans rapidly between the inactive four X,, and the active Form X.+ Transitions ftom Ky, to Xq" occur at @ rate Kl), and (ransiions back secu aa ate Kl Note that Kl) ad k() depend gad level “What isthe aveeage activity, averaged over many transition events betwee X,. and Xo? () “These transitions occur much faster than changes i the methylation level of How ean this te useful in analyzing the model? {0 Solve for the dynamics of A(D) = Xp" following a step ation of attractant Tigand What isthe esponse ine? Mot the dynes sematially (4). Sameas 9 fora step addition of repellent ligand 6conorer 8 Robust Patterning in Development ———_— BI__INTRODUCTION Developments the remarkable process in which a single cel, an egg, becontes a multicel- Jular organisin, During development, the ogg divides many tims to form the ells ofthe embryo. All ofthese eels have the same genome 1fthey all expressed the same proteins, the adult would bea shapeless mass of dential ces. During desslopment, therefore, the progeny ofthe egg cell must assume diferent fates in spatially organized manne to became the various tissues ofthe organism. The diflerence between cells in diferent ts: sues lies in which proteins they express, In tis chapter, we will consider how these spatial patterns can be formed precisely, To form a spatial pattern requires postiona information. This information is carried by gradients of signaling molecules usually proteins) called morphogens. How are mor phogen gradients formed? In the simplest ease, the morphogen is produced at a certain Source position and diffuses into the region tat is tobe patterned, called the field. A con ‘entration pelle is formed, in which the concentration of the morphogen is high near the source and decays with distance from the source. The cells in the fed are oitialy all ‘ential and can sense the morphogen by means of receptors on the cell surface. Mor ‘phogen binds the receptors which i rorm activate signaling pathways in the cll that lead to exprcsson ofa set of genes, Which genes are expressed depends on the concentration of morpliogn. The fate of cell sherefore depends on the morphogen concentration atthe call postion The prototypical model fr morphogen patterning is called the French fag mode (ig tare $1 (Wolpert, 1969; Wolpert etal, 2002). The morphogen concentration M(x) decays swith distance ftom its source at x = 0, Calls that sense an M concentration greater than a Theshold value T, assume fate A. Cells that sense an M lower than T, but higher than a second theeshold, Ty, assume fate B, Fate Ci assusned by cells that sense low morpbogen levels. Mc, "The result isa thre-fepion pattern (Figure 8.1), Real morphogens often fead to patterns with more thon thre different fates,Fon me CHANTTR tam tot FHGURL 11 Merge radeon he Fe age Magge fc Sain gst Ms procestx = Cand dieses ss epoye eaten, reign eda ocean ie at cys th taf the wus at x 8, Cl a he ed ume ate Heese itr than hen fe Wifi cle hele a, nde GAM iw he Fg Ri dpe 3 anne si ut tse thre dines Patterning in three dimensions is olten broken dow cm rok down oedema! ren hah each ans ofthe sues pater by pes morpogen, : Compt aera at ford al at nce Rather tem i sequal roe, Once aia ere ptr fred clin each rin cane ne rovpgin to Bae ner opts Sone pte eae he mere of on Crime morn pdt hs aan tensa sang a te 8 Tet hese st ped tg Ps al ‘out by the developmental transcription networks that we have di chap Aina pees oich ew dsc eng cl monet ote adhesion, further shape tissues in complex organisms. “ Patertng by rpg rails achieved ots achived by dfising myes eed yi chon se si he tin ft ty he pt at iocheml partes A range af experi hus shown ta puter n devel tnt ey robus with espe! ood wre of genetic and errno Gatos (Waingon 199; rn Basso 2000, Wilkin 200; Far et ls 200) ‘he most variable biochemical parameter in many systems is, a6 we have mentioned ttt retin fis Fp sv a cgi et -norhogin production enc very Bl chung inthe sie nd postions of the rus Fr tng csp tin ayn the paring vray changed pom «td eduction tm morph read by stating he mrpgen ye on one fhe wa sie rma tn this chaps, we wi vonsider mechanisns tha cam genes pene hg range a sere tno ae abut to ouch peeturbatons following the twrk of Naar. Bark lb falleagucn (dae etal 2002, 2008, 2004). We wil ett the oat geese patenting seer niens ate not robust Requires ads to spi and tar ek bie chemical iehaniss, 8.2. EXPONENTIA, MORPLOGEN PROTEES ARE NOL ROBUST cet as hepin with te simplest mchanisn, in which mypogen s pred at #suree cated ae ag aul difses ito Bld of identical cll. The morphogen is degeles at rete es We wil see thatthe combination of dlfuson aad degradation leads tan expo ily deaying spatial moenbugen prot The concentration 0° morgen M in ote aul i governed by 2 one-dimensional fusion degradation equation, To tis equation, the diffusion tern, 1 MIO secs to smooth out spatial variations in imoxphogen concentrations. The brger the diffusion Branton De strnge the moathing eect The dsyeadation af moxphogen is described Tpalinca tera Ma eslling ina equation that aes thereof change of M18 ilsion and degrada aMi1=DaMIO—aM wan ty sob this difsion-degradation equation in a given reglon, we need to consider the allesol Mat the bourses ol the regio, The Boundary coutonsare a steady concentra tion of norphogen atts sourceat x= 0, Mi@.=0) = M, and zeroboundary conditions frit heed le) 0, Beene far ito the ik all morphogen moecules have been degra. sa steadystate (9 1d t~ 0) Feqyation 821 becomes a Hnear ordinary diferent equation: Dai Made! a M=0 And the sol Aeyradaton processes jon isan expoentish decay that results from a balance of the dfasion and Mo) Mes @22) “thus: the morphogin lve is highest at the source atx = G, and decays wit distance nto the il The decay is characterized by «decay length N: re NDie 23 “the decay length \ iste typical distance thot « morphogen molecule travel into the Field before tis degréded. Te large the difasion constant D andthe sale he degrada om rate a the lager this stance. The decay is dramatic at distances of 3 and 10. daa cours, the mocpliagen concentration drops (0 about 9% and 510° of Ks inalMy — Fo Be i sg Nt 2 ang sett ah fen rofl ek nnn ante ony eps waa tas op rin sted he por hon ilo) oul ithe Tuts elek by hn se erect one ne re value, Roughly speaking isthe ys Re typical ie ofthe regions that can be pater with such “The fi of ah of the clin he el tee sn heels detriid by theconce tino Eesti i ag wen M ad ha ay ® "n (wo regions occurs when M is equal to T. ‘Ihe position of this bow a given by M(x, = 7, or using Equation 822, Posten ef shoud xis x= Mog (MT) (624) oat have ration fhe mpgs pte tat oof morphogen at he ste M, epoca y ML? fin feet mpegs ‘hat the position ofthe hounds ° the boundary shits x," Mog (94,2, ‘the original nthe sited bounty gure nn Ne thfrene between he B= = 4, =A log (M,/M) 625) hs wal eaton in Mead / ion Mad thi ofthe poston of he Yet by about log 2) 0.7 age shit that vom tod th ose aene atern, Region A in Figuce 8.1 would be aitnost completely lst, eset eat Heo. this type of mecanin doer nt seem to expan the abusnes observed in deelopenl pate «decreases the shift § that occurs upon changes in parameters Semen seroma stele ch the ef mrp 3 INCREASED ROBUSTNESS BY STLF-ENHANCED MORPHOGEN DEGRADATION Fhe ample diffosion and degradation process described above generates an exponential snorphogen gradient tht is not robust tothe moephogen level at its source M- “Fe generate a moce robust mechanism, let us try a more general difusion-degradation process with a onlnear degradation rate FM a Mat = Da" Max = FM) a ‘the boundary conditions are as before, a constant source concenteation, Mb = 0) = ‘Mcand decay #210 far ino the fil, Mo) = 0, This ifuson process has a general rope tha wll aon be sen tobe important for rabies the shift 6 the Moepho- Jen profile upor.a change im Mi uniform in space — it does not depen on position x “That sal regions are shied by the same distance spon a changeit My. “This uniform shif certainly occurs inthe exponential morpiogen profile of the prev ‘us section. The shift in boundary position & described by Equation 8.2.5 does not depend ca Thus, ifsevera regions are patterned by this morphogen, as in Figure 8.1 ali bound tries will be sified by the sime distance 6 morphogen production is perturbed. ‘More general, spatially uniform shifs result wth any degradation Faneion FMD in Equation 8.1 This property is due tothe fact thatthe cells in the le are intally Keni cal (onputterned, and thatthe fl is large (20 morphogen at infinity). "this means that Eqjation #34 governing the morphogen has translational symmetry: the difusion-deg- ratation equations are invariant to a coordinate change x» x + 6 Such shifts only pro- dace changes inthe boundary value atx =O, that i, in M, as illustrated in Figure 8:3 “The spail shit that corresponds to a reduction of M, to Mis given by the postion 6 at which the original profile equals M,’, M(6) = M,' The solution of Equation 83.1 with boundary condition M,” i identical tothe solution with M, shifted tothe left by 8 Gur goals to increase robustness, that, to make the shift 6 as small as posible ypon change in M, to M, To make the shit as small as posible, one most make the decay fate near nO as lege ts posible, so that M," Is reached with only a tiny shift This nk! be done with an exponential profile only by decreasing the decay Jeng A. How: coer decreased comes at an unacceptable cost: the range of the morphogen, and hence the see of the patterns it ean generate, is greatly ence “Thus, we sek profile with both long range and high robustness Such proile showl Ihave two features to provide robustness to variations in M, 1, Rapid decay near x= 2 Slow decay at larg xto provide long range to M. ‘simple solution would be to make M degrade faster near the source far from the source. However, we cannot make the degradation of M explicily depend on sto x hats, we cannot ela = e() in Equation 8.2.1, becuse the cells nthe fle ally ilenical. A epatial dependence of the paranicters would! cequire postions!{CURE 5 cg in martin concerti thes rom M, 9M so ply fem hh moran ae Al oma gulag Mca eftbeshl egal he a {formation that is oot available without prepacteraing the field. Ove only recourse nonlinear, selfenanced degradation a feedback mechanism that makes the degradation rate of M increase with the concentration of M, A simple meade! for se-enhanced degradation employs degradation rate that increases polynomially with M, for example, AMit~ DaEM/Ax ~aMe (32) This equation describes a nonlinear degradation rate that is large wh tion is high, and small when M concentration i lowe! A steady state (2.M/I L= 0), the moephogen profile that solves Equation 8.3.2 is not ‘exponential, hut rather a power la MoAb HOt en @Mlen” aod a9) is power-law profile of morphogen hes a very Tong range compared to exponential roi To obtain robust, long-range patleros, iis sullicent to make M, very large, so {hat the parameter € in Equation 8.33 is muh sinalee than the patter size (noe hat = 11 JM) this iit the morphogen profile in the ld does not depend os Meat all . {a mrp Aan ton fas porate sg sy sewn fc xa las item te el metn eee nate thergaroltceme ocean) BE Rates pee fis tons “ ® a » GUS 14 Comparison er be pis a A fle orgs at Feats olinca dat sos expotl ptt aly ae Gli het Abel pe ‘Gate Tn) wa stage y wn be mop ths Hor, My aa Te eg Incl fae boondry (> cma othe tce AX fen tw bai ate pete pee iss deed ny the pins tc the pte some hes iby the ral ltl es Nate IRs guia sea 0 Wien he mang undrsna walinca enh Seaton, poe topes pa b enisinil of ealy ate fo ence Bs giao ha Tess rete sane in Gao gua gion we (Egon 832m Ett 208) Me Ant ea s0 that there ace neigible shifs even vpon large perturbations in M,, Patterning very robust to variations in M,, as long a5 M, does not become too seal (Figur 84). The power-law pratile is not robust fo changes inthe parameter A ~ Dia, the cai of the dlfsion and degradation rates: However, parameters such as difsion constas and spocific degradation rates usualy waxy much less than production rats of poten sich athe morphogen. Ta surmesaey,selEenhanced degradation allows a steady-state monphogen pole with a nonuniform decay ete, The profile decays rapidly near the source, proving robustness to changes in morptogen production. Ie decays slowly fr from the source, allowing lang ranged patterning, f4— NETWORK MONS THAT PROVIDE DEGRADATION FEELIACK FOR ROBUST PATTERNING ‘We saw that robust Iong-range patterning can be achieved using feedback in which the :morphogen enhancs is oven degradation rate. Morphogens theoughout the developmen: tal processes of mary species participate in certain network inti that can provide this, selRenhanced degredation. ‘The robustness gained by selienbanced dageadtion might cexplin why these rulatory patterns are so common, ‘Use morphogen M is usualy sensed by a receptor Ron the surface ofthe cls inthe field, When M binds Rs activates sigaal transduction pathway that eds to changes in gene expression. Two types of feedback loops are founel throughout diverse devdopsne tal processes (Figure 85). EESo o G58 5 to tm ai at pode seesinced gestion Hf ephagen ML) Mi ‘eceper Rand ctats signaling acy inns Meeps Shanta Re skenop yale ae Geadoeros and Belgrade (6 Mates sigoling potas tht eps Kees Taree Rhus and tits am exter pa pretest dgrads Nam tis ces aoe Skrstaton. tbh and, aero drain ate The frst motif is a feedback lap in which 1 EM An exsmple i receptor R-enbances the degradation 1 morphogen M = Hedgehog aul ks receptor R = Pete, which Participate in patterning the fruit fly and many other organisms, Morphogen binding to "triggers signaling tht leads to an increase inthe expression of R. Degradation of Ms > Dy). Fett iermore in the ‘oust model, eee Tis not degraded bythe protease P. In fc, P ean only degra within the complex C (a, > aj). The robust mechanism is well described by the following set of steady-state equations, setting ime devivatives to uero, hey are simpler than the full ‘eatations because they have (wo paramscers st 10 2em0 (Dy = 0, a, = n was concentrated DP O2-kIM<0=a000 (5a) De 2! CIP HKIM a PE=0= 2008 (855) TRIM Fa PC- 0p MMae a0 Remarkably these nonineas equations can be solved anata Summing Raustions 8.5: and 8.56 shows that C obeys. simple equation bea Cae sn ‘Te general solution ofthis equation is CX)» ax + Cy bu det the symmetry ofthe Problem in which the lft ond right sides of the DR are equivalent, the anly solution is spatially uniform conceauiation ofthe comple CO) const-¢, (as9 Using this in Equation 856, we find that the product of Fee fan M is spatially uniforms KIM= 0.9, 659) sod therefore Equation 8.4 can be witten explicitly for M, using the relation between T fd M from Equation 859, to finda simple equation for 1/Mt BM Ve = kD, (ss.0) vwhoe sation fea function peaked near Moe) = Alte? 8) AS? Pyle san ‘he only dependence of the morphogen profile onthe total levels of M, Mia. 8 through the par c= AMay asia “te uae cinema very aly ang te laos of po Na sunt ine. Inthiscase the morphogenic becomes x power aw stot dpennon any fhe paramere model ep A= 20), SMa) fafrom ming 33> asin ne described by thi equation tn prt he ee MOO ple yf he mi cin suerte eto ot rhs stayed on et Cfhcptene orator dnt pear hescin Sra Inauramary th ce morpogen pole robust othe vel fal potest Shc ander gener long ange pte tos power des. iow des tha rmechnigm wrk The mechanism raed on shating of morhogen oythe nine Morgen ane ve ns snl ina he DRY ce “lng th iter Once comp eed he moro erste Inconvenient ol Tt ct he bn of th Dy agen Tn pied the Dandscamistes where conentrton as owes theming re inhibitor Sw lei nd omc speed eer hy egal Pere tie the itso pret he mine bonnie ey Ts br oot ay cite re a Sy pa waned day ten a e paei srry hereienent long rane us patering hat we dane a cS Re Mateo 83 ts done wate eying ah trechaisms ado longanged pow rls, “enema ee wo por ox ti mend sore The at nhs 1 ead nly whe ope My an nt whefie, ‘he second is that M cannot dfs unless bound o Hoth ofthese properties have been dewonstrated experioventaly, dhe latter following the theoretical prelicion (Fhlar etal, 2002) Move generally this chapter and the previous ase aimed to point out tat robustness ‘an help to distinguish between dlrent mechanisms, andl point to unexpected des Only a small fraetion of the desigas that generate a given pattern ean do $0 robust Therefore, the principle of robustness can help us 1 ative at biologically plausible mech anisms, Furthermore, the robust designs seem lo show a pleasing simpli FURTHILR READING. Berg, H.C. {1993} Radom Walls Big Princeton Universe Pres, Eldar, A Dart, Ky, Weis, D, Ashe 1 Shi, B.Z, and Bata, N (2002), Rubuastnes ofthe [BMP anorphogen grader in Prop embryonic patterning, Natur 19 304-308 ilar A. Rosin, D, Sil, BZ, and Berka N. 200, Sell enhanced ga dealt undesie robustness of moepogen gradients. Dev. Cel 5635-086, lidar A Shilo, BZ, and Barkai, N- (2004), Ehcdating pecans wider ‘nurphogen gradients. Cure Opin Gee De, 438-439. tg rdbuniness Aectional Reading Kirslner. MW. and Gerhart, LC. @0US) he aust of ff, Yale Unverily Press, Lawrence PA, (998) Te first coordinates [a The taking af ye The Genetics of Animal Design lucha Science 1. Chap. 2 Slack, 1M. (1990. rom ig 10 Embry, Combrilge Univesity Press, UK. Chap 3 Wolpert, 1 (1969. Positional itormation and the spall pattern of ella dierentiain. J ‘cor Mia, 28:17 8.1 Diffusion from both sides. A moephogen tx produced at both boundaries ofa region of cells that ranges from x = 0 to x= L. The morphogen diffuses into the region and is degraded at rate a, What is the steady-state concentration of the morphogen as ‘function of postion? Assume that the concentration at she beundaties iy MC) = 'M(l) = M,, Under what conditions i the concentration af morphogem atthe center «of the region very small compared to M, Hint: ‘the morphagen eocentestion obeys the difusion degradation equation st steady-state DeMi?-aM=0 ‘he solutions ofthis equation ate ofthe form: Moo ee Bowt aa, 84 Find A, A, und What satisfy the difusion-degradation cqeation and the bound ary conditions Digfusion wih drgradation at bourulary. A morpoges is produced at x = O and centers opionot cells wheteit ie not degraded, The moephogen is, however, strongly degraded atthe other end ofthe tegion, at x= L, such that every molecule of ME that reaches x = Lis iimdiately degraded. The boundary conditions are thus MO) = Mand ML 44. What isthe steady-state concenteation profile of M? bb Is patteming by this mcharism robost to changes of the eonceatration at the soc, M(Q) = M,? Hint the morphogen obeys simple equation a steal state DEMMx=0 “Try solutions of the form M(a) = A x + By und fi A and Buc that Mix = L) = and Mix = 0) = My Next, ind the position where M(x equals a theeshold nd find the chaagesin this postion upon a change of M,, Polynomial sef enhanced degradation, Find the steady-state concentration profile of ‘a morphogen prosiced at x= 0, The morphogen dilfuss io a fed of cells, with nonlinear self enhanced degradation described by aMi1=DI'MGxt~aM ‘When is patterning with this profile robust othe level of M atthe boundary M2 (x + by" and find the parameters a and b in Hint Tey a slation of the form Mi) terms of D, My and a Rolhust timing & signaling protein X inhibits pathway Y. ALtime t= 0, X production stops and its concentration decays due to degradation ‘Ihe pathway ¥ is activated when X levels drop below a theesbold T. ‘The tise at which Y is activate ist Our ita lowe of X X40) =X gli to abe ty a robust as possible to a. Compare the robustness of ty in two mechanisms, linear degradation and sll: ‘enhanced degradation (note that inthis problem all cancenteations are spatially uniform), axar=-aXx axjate-aX"4s, Which mechanism is more robust flctations in X,? Fsplain, (> Hapa why robust ining mechanism requires a rapid decay FX at times close woino, Activator acculation vs, repressor decay (ha 0 eay (harder problem). Compare the robust ess of in problem 84 to an alternative system, in whieh X em attr tat igins to be produced att = 0, activating V when it exceeds theesild T. Consider both lineae ar nonlinear degradation of X. 1s the accumulating tet nism more a ess robust to the rods ate of X thin the de tor mech ving repressor 86, An action whats in generally eos rubust 10 variations in the prod arations ithe production ote of X than the decaying repressor mechanism of problem 8.4. (Rapps al, 2005). “vs ex ondary condo: Merphagen ghogen Ms rode atx =U and dine nto a In fi ofc wie dept atte Soe rss pe sing bose conn ae fa) a'x va] aDdMIOe oma the solution discussed in the text, which used a const rh " thes tant concentraion of M atx ~ cnarrer 9 Kinetic Proofreading 93 INTRODUCTION, oe In the preceding two chapters we have discussed how circuits can be designed to be ‘robust with respect to fluctuations in their biochemical parameters. Here, we will extn ine robusiness to a different, fundamental source of errors in cells. These errors result from the presence, for each molecule X, of many chermclly similar molecules that can confound the specific recognition of X by its interaction partners, Hence, we will examine the problem of molecular recognition of a target despite the background of similar mol- eciles, Hove cam a biochemical recognition system pic out a specie molecule in asea of other molecules that ind it with ony slightly weaker affinity? 1 this chaptes, we will see that diverse molecular recogaition systems inthe cell stem to employ the same principle 0 achieve high precision, This principle is called Kinetic proofreading. The explanation ofthe structure and funetion of kinetic proofreading was presented by John Hopfield (1974) “To describe kinetic proofreading, we will begin with recognition in information-tich processes in the cll, sch asthe eeading of the genetic code during translation In these processes a chain i symthesized by adding at each step one of several types of monomers. ‘Which monomer is added 3¢ each step to the elongating chain is determined seording to information encoded in a template (mRNA inthe ease of transtation). Due to thermal ise, an sicartect monomer is sometimes alded, esting in errors, Kinetic proofread sng isa general way to reduce the errr rate t levels that are far fower than those achiev: able by simple equilibrivm discrimination between the monomers [Aer describing proofreading im translation, we wil consider this mechanism fn the context of @ recognition problem in the imeane system (MeKeithan, 1995; Goldstein t ah, 2004), We will see how the immune system can recognize protein that come fom a dangerous microbe despite the presence of very similar proteins made by the healy boul. Kinetic proofreading can use a small difference in afinity of protein ligands (0 Bsmake a very prose decision, protecting the body fon attaching sel Finally, we will discuss kinetic proofeeaing in other systems kKincic proofreading it a 30 vwhat subtle ides, ara! so ye wall ase the dierent syproaches to describe i. tn the context of recognition in translation, we will use kinetic {equations to eve the error rate ta he context of the immune recgnition, we will oes lay time argument Bul first we wll tll story about a cognition problem ina muscu Avan anatogy to kinetic proofeading, conser a mascors cater who wants to design 2 coun that would elect Piasso lovers irom among the nseum visitors. In this muscu, Hal of the vistors ate cast fovers and half donot care for Pieaso. The carator opens 3 dour in a busy corridos. The door leads to 3 toom with « Picasso painting allowing Visitors to enter the room al andlor, Pleasso lovers that happen to ent the room hover near the pictue for, on average, 10 min, whereas others stayin the room for way 1 min. Because of the high afinity of Picasso lowers forthe painting, the room becomes enriched with 10 times more Picasso lovers than nonkives, ‘The curator wishes to do even better, Ata certain moment, the curator locks the our to the room and reveals a secoud, one-way reveling doot ‘the aunlovers in the ron leave through the one-way dsr, and afer seveial mi Picasso lovers, tll hovering around the painting. higher than 10-ola the revolving door were two-way allowing visitors to enter the room at random, nly 4 (0-fold enrichment for Picasso lovers would again occur. Kinetic proofreading mimi ‘he Picasso room stratagers by using nearly ieeversible, nonequilibrium react way doors es, the only ones emai are ichmien for Piasso lovers is mich 9.2 KINETIC PROOIREADING OF TH GENETIC CODE CAN. __ RLDUCE ERROR RATES OF MOLECULAR RECOGNITION Consider the fundamental biological process of translation. in translation, a ribosome produces a protein by linking amino acids one by ane into a ehaie (Figure 91). The type ‘of amino acid adied at cach step tothe elongating chain is determined by the information «encoded by an miRNA. Fach of the twenty mine acid is encoded by a eadon, a series of three letters on the mRNA. ‘he mapping between the 64 cals ad the 20 amin acids ‘scale the genetic code (Cigure 92). To make the protein, the cuion must be eead and the eoresponuling amine acid mont be brought into the ribosome, Each amino acid is brought ito the ribosome connected toaspecifie RNA molecule. That (RNA bas a thet recognition site tha is conple- ‘mentary, and pais withthe codon sequence for that amino aid on the miINA (Figure 9.0. There is RNA for each ofthe codons that specify abn aids in the genetic code, ‘Translation therefore communicates information from mRNA cadons to the senine seis in the protein sequence. The codon must recogaize and bb ke correct RNA. and ‘oot bind (© the wrong CRNA, Since this is @ molecular process working under theemal noises it has un error rate The wrong tRNA can attach tthe codon, eeslting in a trane- ‘avo error wisere a wrong amino cit is icoeporated in the translated protein. These ‘eansltion errors uccue at a frequency of about 10" This means that atypical protein of ko 1 me of prot he ue he MRNA sc by RNAS tha psy eps of eterno te vt Irrals the cendon, she arin ack shat it peso ar he Rh cd ods When RNA ke theca i “testminin tr tc pei et a sie iets hn iN th na a ah A oe {EEN ber He pene sl rt RA ah 7 wrest st ons RNA sje | a | ia | eps) el mi. 3° Lew Pro, Gn ang ag Peet etot ates “ ie THe us ag [A . |p Ss val a Gu Gy |A fhe} e] 2 fs to aio acldshasa chaps hve oe wong amin wil. mh ihe trae would be disastrous, becuse it would result the malfunction of an unacceptable fac tion ofthe cll’ proteins 9.2.1 quills Hing Cano Explain the Proeision of Hanslarn “ gots th te spt na rhs ecgaton pcs seam ining Sido Wea wth Sp livin ning ona xe he ee"reo Fate Us fy boeause equilibrium binding Reneratesceror rates that ace equal to the ‘tio ofalinites ofthe correct and incorrect IRNAS. This wou result in ere rates that ste about 100 tines igher than the observed ereor este “To analyze equilibrium binding, consider codon Can the mRNA inthe ribosome that ‘ncoukes the aman aci a healed t the protein chat, We hegin with the rate of bind fing ofthe correct tRNA, denoted c, to cdion C. Codon C binds e with an on-rate ke "ANA uniinds from the codon with otra. When the RNAs bound there is probs bility ¥ pec unit time that the amino aed attached tothe RNA will covalently inked { the growing, translated protein chain, tn this cas, the frced URNA unkind from she «aon andthe eibosomeshifis to the nest codon inthe mRNA. The eilibeiom process Ishonee 4 CeesfeC]—Seorrect amino aid oan ‘AL equilibrium, the concenteatin of he complex [6] is given by the balanee nf the ‘to arrows marked k, and k,” (the rate v is much smaller than k and kad ean be ‘eglecte). Hence, at steady sat, collisions of cand C that form the complex eC) trite balance the dissociation ofthe complex je], so that eC k= [eC] k." This tests in & Kd Benes By "ernst st el of a ofc and re ni sncpeatin he ere te ppeuimasy ep othe ato of the scat cm Sant ng she concent (IN connate shout te sine fr ea Fy = Raf Ramne =¥d CREE Ky KK 027) To repeat the main conlson the ero ra equim ogi stern bythe ratio of dissociation constant for the correct and incorrect (RNAS. AS o> k, because of the weaker clemica! bonds that hold itm the bound com- fon 92.3, we find ples. Using Pa e238) Rega ® KAR y= hl ‘he off rates are akin to the dittocation rates of museum visitors from the Picasso paint {ng inthe Piasso room story above, sernatinty ow does equim option compare wit bat err te he ait cons eta inarest RNA ms exit meso tS an af Fatio of about K/Ky~ 1/100, Henee, there isa large discrepancy between theYeu m CArLLK ‘equibrium recognition error ¥ » KJK,~ 1/100, andthe actual translation ero rae, > 10,000. t therfore seems that equilibrium recognition canoox expla the high i ity found in this system! YA Rivets Panoading Can Dnata ede the bo ‘We just saw dat equilibrium binding ean only provide discrimination that i a good ay of the correct and incorrect targets. Wh micas can explain the high felity ofthe translation machinery, which ia unde hi than predicted rom equilbrivms secognition? the ratio of the elma a hee “The solution les in a rexctin that occurs i the translation process whic: was well kknoven at the time that Hopfield analyzed the system, bul whose Fanction was not wider stood ard was considered a wistful side reaction. In this reetion, the (RNA, after Toning the codon, undergoes a chemical modification, Tat Is « binds co C and then i converted toc, "Ihis reaction is virtually iceveesible, because its coupled to the hyo. sis ofa GLP spoecule2 he modified (RNA, c, can ether fll ofFof the codon or donate its aminoacid tothe elongating protein csi: —eoreect amino acd 029) ‘The fact thatthe modified RNA. can fal olf seems wasteful because the costect RNA ‘ean lost. However itis precisely this design that generates high fidelity. "Ihe secre is that © lls a secon lisrinsnation step: the weong (RNA, ence modified, can fll of ofthe edn, but i cannot mount back on. This irteversible reaction aets a the one-way dae in "he Picasso sory “To compute the error rate in this process, we need to find the concentration of the ‘odtfed bound complex. The concentration of fe"C] is given by the balance of the to processes described by the arrows murked with the rates mand I (ince the rate is ‘ca smaller than the other rate) leading to balance a sealy sate hetwcen mice: tion ofthe complex eC] and the dissociation of e* a rate I’, mfeC] = let, yeding 9 ‘aesly sate eatin: ec] =m ev 92.10) et ret NAS rove dr tie den ucarct elon eg nh Sin Jc hie Ny stv mol tne no ck cea gee inna tt he cae rose (nenne 934 Th ‘hth high siya sw caps eee Tyce ner yen ectio o 4 teeming ean AT seep con ty fot ‘Nostale eoandceney praise 9 sn tha expt fe py a se amis the linking rte v times the moulfed comsples con ‘The cate of correct Hacorporn ceoteation (Equation 92.4 Roar VIER = MECH 024) capes ord Teanveson fo uous athens Theses es races sm dite etn NA enor toca sn He ate thn allo he on homee ch Tae hat he di cane tne hea aio the wrong RNA the Te geafiy af tcc NA The frat ol cone con ee the ne era fr the not ARNAS, se they areal ognize by the same eon Wythe = thy = Kul 9242) ination ste, with 2 significant chance that the “thos, d undergoes a second discrimination step, hac song itNA iremored The rat of wrong amino ci linkage ite same ss 9 Ea Joost th all paranctes for € replaced th de corzenpoding parameters or Bang =¥ RCH = ¥ GI Ry 0219) resulting in am eso ate, using Equation 9.2.12 Fe Ra Baer = KIKI UTIND = RSI? = Be oan) ee ni neo 0219) al ects been revere and teu, no jon 9h the ee mera be ped over th me eens as : ia Gata nena 92 heer sel with died Gell Bt a paso rom in whl th one ori changs 9 a0 ay Se ne cce Poe a elm cognition rato abot f= 14000 8 mich ove ee ee unn00 snr othe bsered er ate . Foe altel can be aed by king gsr 9 reveal cad orl protelig Pees Its important to te thats801 RECOGNIZING StL F ANI We have just seen how kinetic in translation. We will now use as proofreading, base on time dela ‘he immune system monitors the pathogen, the immune system comptes and mobilizes the a nmnune system is made of vast col inyziad ways, ‘One ofthe major tool ofthe immune system is Aesigned to bind with high affinity to the antigen, ‘One ofthe important roles of the imm antigens, for example, for proteins ning takis caried out by Tcells. Each ody against a fvsign protein on heer ay bo ial eeprom ce el Misc comple arena tee ele =o) ein of Gre ppt igen. Ta peovide (9216) i overall error eat of 2.7) DNON-SILE BY THE IMMUNE SySTEM Proofreading uses 3 nonequilio ilviury step to rete errors ihlty diferent (bat equivalent way to explain kinetic ys or this purpose, ml sya ofan psa the mung sean nn M8 ll body for dangerous pathogens. When it detects op epone e lection of cells that communicate id i. antibodies. Hach antibody isa protein specific foreign protein male hy pathogens called system isto san the cells of the body for i by a vu thot has infected the cell The sea ‘of the Tels has receptors ie ofa specifi anti Formation for the T-cells, each cell in serge » ets Tage cells potent gna thle pee i Teal can egnie specie cg pts ea sities Mela nep the body presents fraginents of proteins onthe cll surface, he proteins are presented in edicated protein complexes on the cell surface called MHCs (Fgue 8.3}. “The goa of the T-cell st eliminate infected cells, Gach T-cell ean recoguizea specific antigen inthe MFC because ts receptor cam bind that Foreign peptide, Ifthe Tel ree tor recognizes ts antigen, the foreign protein fragment inthe MHC on a cel triggers a signa transduction cascade inside the cell. The signaling causes the vl to ill the cell that presented the forsign peptide. This eliminates the infected cll and protects the by from the virus. 1m the recogoiion proces, itis ecsential that the Teel! des not lel that present proteins that ae normally produced bythe healthy body. [seh mriseeogniton accuse Smaune sytem attaks the cells ofthe boy, potentially lading to an autaimmune disease The precision of the recoguition of non-selt proteins by T-cells s remarkable. Fels «an recognize minate amounts ofa foreign protein antigen in a background of self pro: teins, even though the sel(proteins have only a slightly lower ality to the Tel receptor than the forsign target. The ereor rate of recognition is fess than 10, slthongh the alfinity ofthe antgen is often only 10fold higher than the affinities ofthe self proteins 441 Lquilariae binding, Cannot Explain be Low Fear Rate cof Temnune Recognition The receptors on a given Tecell ate bull to recognize a specific foreign protein, which we will ell the correct ligand, e, The correct ligand binds te receptors with high finity. In addition tothe receptors are exposed to 2 variety of self proteins which ind the recep- tor with » weaker affinity. In particular, some ofthese self proteins are quite similar to ‘the corect ligand and pose the highest danger for misrecogoition, in which the receptors mistake a selprotein forthe correct ligand. For cavity let us teat these wrong ligands 45 8 single entity d, with a lower affinity to the receptor. We will begin by the simplest, model for recognition, ka which c and d bin! the receptor in an equilibrium process. As in the previous section, this yields ezzor rates that are proportional tothe rain of afin ties ofthe incorrect ane! cortect targets Since the ants ofthe correct and incorrect ligands are not very different, equilibrium eecognition results jn an unacceptably high eof latecogaition, ‘The dynamics of binding f the correct liane to the receptor Winches two process te first process i olisions of e and Rats rate k,, (0 form a complex, fh in which the Jigand is bound to the receptor The inverse proces is dissociation ofthe comple, in which the ligand unbinds form the ecceptor a rate Ky. The ate of eange ofthe concentration of ‘hound receptor i the diference between the collision and dissociation rats: eRIt = hye Rye fe) Al stood state, dleRdt = O and we find leks ROK, ‘white K, isthe dissociation constant ofthe corret ligand to the receptionWhen the Hyand diods the eeptar, it wiggers a signal transsction pathvray inside the Tel, whieh leads to activation ofthe cel, Once liga binds the receptor, the sig a ¥ per unit tine, Therelre, the rte of Peel nctivation in the presence ofa eancenteation ¢ of corte ligands 1 pathway activate with probabil Ana [ER] Y= eR WR, Asis ‘thew staf equations deserbe the biaing ofthe incorrect ligand d to the recep, ate and wiErate ofthe incorrect ligand ae kad Ky ealing alate = ki A RK I he steady-state concentration ofthe incorrect comples, [Ral is given by the proauct of ‘he concentration of and R divided by the dissociation constant fr Ike} = Ra, where Ky Kgl ‘The affinity ofthe incorccet ligand is smaller than that of the correct ligand, so that X,> K. As mentioned in the previous section, ths difference i alinites i usualy dae to the difference inthe offrates ofthe ligands, rather than to different on-rates, The cor fect ligand dissociates fom the receptor ata slawer rate thas the incorect ligand de to ity stronger chemical bonus with the receptor, Kyr< ky. I ether words, the correct Ligand spends more time Bound tothe rezeptor than the ineorcct ligand, {nthe equilibrium recogaition process, wisen the incortect ligond binds, it can cl vate te signaling pathway in the Tell withthe same intense probability ss the correct ligand, . In equilibrium recognition, the receptor has no way of distinguishing between ‘he ligands other than their aisites. "The resulting rate of activation due tothe binding ofthe incoreec ligand i Arm HIR] yd Rw, Hove, theresa the Toeele defined bythe eatia of inerrect to sores activations Fy= Anaadoune Kd R K€ Rv (KK) Ee) Te eror eaten this equilibrivesreeogeition process is thus given bythe ratio of afin ies of the incoreeet aud corcect lguads, times the ratio of thei concentrations fa the ‘immune system, the incoreect ligands often have only a 10-fold lower alfnity than the correct ligand, K,JK, ~ 0.1, Furthermore, the canceateation of incorrect ligind (proteins ‘made by Ue healthy boy) aften exceeds the cancenttation ofthe correct ligand (pathogen protein). Hence, the euilibrium ereoreate is F,> 0.1. Ths far higher tha error rate in'T cell cognition, which ean be F= 10° or for. he observed GUE 14 Xtc padng model in Tc spor igang inert tthe ‘Sr tna ene eg pata a ad he serie pcan cia tm (expos eign reine bs of il a the nao see ees Winsitewtnecetes a dl betwee and gad gating Only ands it remain ond tought bday sn tease How can we brdge the huge gap between the hiyh rate of equi ‘errors and the observed low ereor rate (othe real sytem? The next section describes & kinetic proofeadiag mechanism in the receptors that amplifies small differences in alin Inyo lange dferoces in the recognition rates. ners Filly of T-Cell Recogy 942 Kinetic Piofreslng a ‘thea gh proces inl eile er ans tp hh tray at Het agi spent ob serine dal, Alor banding the por alps sets fol mean sch pooporyaon on muerte ge. thteodieaton ae ee consuming and te ld ayn era eee moe. the cer binds saa pon parts ie the ‘xin othe tgrling pty nie hel ino afer alo ese nol aanee shy tundrgenens are compl, Ries prose reson hese x Sep weer aay ethno these solace eo ete he bebe en tht Sai iu tema ound othe rcpt or ng cmgh tea aes 6 Eat Pea an 935 “a cdrton ns let seus binding evn fhe ots nd Oe oun thc gan tas pty per oni ney deter the epor enc he sty temas bound for ane gran taer ining Pee 2h Signaling in the cell only occurs at a delay rater ligand binds the receptor, due tothe sores of modifiesions ofthe receptors that is needed to activate the signaling pathway Hence, the probability pe ligand binding that te T-cells activated i egal 1 the prob- bility that the lind fs bound for a ve longer thaSimilarly the incorrect ligand has an fzate Ki. The orate of the horse Higa is ay mentioned above, larger than that of the corset liga, Because i binds the receptor wore weakly. The probability tha the incorrect liga setvates the recepkris Hence, te rar rate i the delay mechanisos i the rato ofthese activation rates: Al ‘This allows a very small ervor rate even for moderate differences between the offraes, provid thar the delay is long enough (+> ky), For exemple if the afte of the correct ligand iskg= 1 sec" and the incorrees ligand i k= 1D 60“ andthe day is ‘one finds Sct, ne 10# ‘Thus, long delays can enhance fidelity. However, this cones at a cost, "The longer the Aelay the larger the numberof binding evens ofthe carect ligand that unbind before signaling can begin. Ths, increasing the delay can cause ass of sensitivity. The lss of sensitivity is tolerated because ofthe greatly improved dsstioination between the cortet ligand an incorreet-but-chemscsly-sniar ligands Kinetic proofreading is general mechanism that provides specifety due to 9 delay ‘ep that gives the incorrect figandls a chance to dissociate before recognition is compete In order for kinetic proofreading to work ellecivaly, the receptors must se their mod fictions when the ligand wnbinds before new ligand molec can bid Otherwise, the ‘wrong ligand! can bind to receptors that have some of the modifications from a previous binding event, resulting ina higher probability for misrecogotion, "Experiment to test kinetic prootreading use series of Hands with diferent Kyyvalies (covieweat fn Goldstein etal, 2004). The experiments ate desigael so tat the fraction ‘ofthe receptors boul hy eal ligand is the same, This is achieved by using higher con centrations of ligands with weaker binding (larger ks or by norman the eesults per binding event. Simple equilibrium recognition predicts @ cnnstant probability for tig ering signaling per ligand binding event, egos of the ky ofthe ligand, In contrast, tine experiments show thatthe probability of activation of the signaling puthway depends inversely on ke Thismeans thatthe longer the ligand is Bound to the receptor, the higher the probability that it triggers signaling, This is consistent with the kinetic proofreading picture Kinetic proofread se8 modification of the T-cell receptor afer ligand binding to create a delay. This process is not unique to T-cell ceptors. Infact, these types af mod fications occur in practically every receptor in mamivaian cll, including receptors that sense hormones, growth factors, nd other ligands. This eases the possiblity that delays and kinotic proofreading are widely employed by receptors 1 incense the flity of recognition. Kinetic proofreading can provide eabstness agains misrvognition of the tackgrouod of diverse molecules i the organism, 9.4 _ KINETIC PROOIRCADING MAY OCCUR IN DIVERSE RECOGNITION PROCESSES IN THE CFL “The hallmark of kinetic proofreading isthe existence of nonequilibeium reaction i the cognition process that frans an intermodiate stale, providing delay ater ligand bind Jing. The systems mast operate say from equilibrium, so that ligands cannot crcunvent the delay by rebinding directly in the modified state. New ligand binding mast rimacily wu he ute tte “These ingredients are Found in diverse recognition peoceses in the cll An example is DNA binging by repair proteins (Reardon and Sancar, 2001} and recombination proteins (lusty etal, 2004), One seh proces is responsible for repising DNA with a damaged base-pair, A recognition protein A binds the damaged staal, becsse it has a higher affinity to damaged DNA than to normal DNA. After binding. protein A wndergoes a modification (phosphorylation). Wher: phosphorylated, it recruits aiional proteins B 1nd C that niek the BNA on bath sides of A and remove the damaged strand, allow: fing specialized enzymes 1 fill the gap and polymerize a fees segment in place ofthe damaged strand, The modification step of protein A may help prevent misrecgniton of normal DNA as damaged. ‘An additional example occurs ia the binding of avino ac 1o their pee RNAS (Hopfield, 1974; Hopfield etl, 1976), A special enzyme recognizes the RNA and its spe: cific amin acid and covatenly Joins them. Covalent joining ofthe wrong sine asd to the &RNA would lead to the incorporation ofthe wrong amino acid inthe translated protein, Interestingly, the eror rate in the TINA formation proces i bot 10+ snilor to the translation error rate we examined in Section 92 due to emisecognition betes RNAs and their codons. This low ertoe rate is achieved hy aa intermediate high-energy sta, in which the enayme that connects the amino acd tothe RNA fist bide bath teactats, shen modifies the RNA, and only the forns the fy and {> Ihis leads to the following inequatities pe tadeand p> site) regulation selected (3.41 Similarly the uns when f ted design in which Z:is eon 11,2 leading to she inequalities itatvelyexpressal will be selected p>efband p> 1 1ie 939) these inequalities qutions 10.4 and 10.35) linka property ofthe cavironment, the lenis for 7 lined the faction of He p Una contin C, accu, to the est al ‘benefit parameters of protein Zand its regulatory system, For each valucofp, these eqs ‘ions tel us whether eegulation willbe selected over simpler designs ‘The range of environments in which each ofthe three designs is opti is showa in igure 10.6, Regulation is sclected at an intermediate range of demane,p- Hig demand tends to favor systems that are continually expressed, "The design where Z is continually ‘expressed is always optimal when p= L because if Zi lways aceded, regulation becomes superfluous. When p = 0, the protein is never needed and the optional mechanism i 10 never expaess ‘The three dorsains in jar 10.6 meet at & point. This point bas coundinates p* = fb (ile © 1 ~ fb, The targr the cost af producing the protein, e,relalive tits bent, 5, the more this point, which corresponds tothe apex ofthe triangular region is Figere 116, moves to the right and down. When costs exceed befits, > this region vanishes stu regulation is never selected In fat, when e >b, selection favors organisms that lack Z altogether, because is cost exceeds is benefit. The smaller the ratio ef peotin cost to benefit cb, the larger the range of environments in which regulation is lected ‘here exist organisms in nature whose envitonment is quite constant. An example is Intracelllar prasites, organisms tht lve within cells and ave supplied with nuteents snd stable conditions (Maran, 2002; Werneyree, 2002; Moran, 2003, Wicox etal, 2003), oven evely protein bas ether = Loe p= 0 lese orgasms Indeed lose must of their regulation systems, seh as transcription fuels. "They hold & small of yenes continually expressel and ack many ofthe genes found in elated, 0 Parasitic organisms. this agrees with the behavior shown in Figure 10. 0n the lines p = and p At the other extreme are bacteria that live ia constantly changing and challenging cavironments such a6 the soil, These organisms have compratvey large genomes dense ‘ith proifieregultion systems? These Bacteria probably have « p< 1 lay mast genes, ‘hat extensive regulation fs selected as shown in Figure 106 "tena mb af vamp Str tt sine wih th mer frie ew neem annem when ecm iia toveie mtn nts npn ene OP MAL Gane Ginees Spe syste suc An 0 Secon te agra tigen wee ane jays arp texans ithe tan tne tata sow Drotin Zinta provider elt he eal Theo pee cet In sumary,eplton makes sce only if te eionent ace te var eenment te ost a he ain sie ot _ infomation processing tht can respond changes the eBHIMETE yee Wehoe ths examined the section of the epson eve! of EDS gh tion of gue regulation systems. Cost-beefit analyses us 299 forces that drive these evoltioary process A nal example tm contenft analysis of gene iret the fedora oop neta code 10.4 ENVIRONMENTAL SELECTION OF THE HLED-TORWARP HOOP NETWORK MOM ag ‘AS we have see throughout his ook ene relation storks on Fy cone mentary circuits termed network motifs. Evolution appears 1 hae nen verge on thee mois in ferent eras ab wl in eet SET oy 0 understand, in a highly simple mode, under which environments ‘ular nti might be selected seth Forth juror well sami onthe wt immon ek A a cet aoa loop (EDs we sain Ctr the eshte FS base nami funtion: It abode ing ON eps a2 PNET ge fe OFF tp ‘The FP i weed eansrptn newark a of inde an PL nthe ol ansrgion networks fo exe Se ed PY non gnes regulated byt inputs are elt by an FF, and 6% 8 imple tint eign (oth types of rete are shown in gare OD) intresting to ask why the FFL s selected nsomesystems and ot ONY ar te “answer tht question, ft prom «simpli antes ST ple selection ofthis ger cect in a ven dynamily Matai EOE? gal in sly 208 hy eronent ean th depen pies oF em the natural bia of the organs, We wil rd conitins that the ——eG) thet can be acted creme nate debut pat Ped to ste en ad conan bth ang aso pts New dere theopinl ete dy of he cea fon a econ sensed eno 10 1009 Tema reef hess aes allo Sep postition etd ithe ener dan The ns oe Centon tc serch pein Zan bina oe hep ton SO) cf At tap etn tsar ase np ae Thon fi on go gre 08 se ea oa ecto Tees tht tht uc rsp ero Piste dain Sie nec by exresion pin Zn regan oben pls 2 el scan eo eps to bit pla alow ns on ope Daten cn b adrnagnt Ae we so Chap coherent Fen perm lay ie pet Hsing Tn he tet pes FF, Zaps na Sal sn th sate sph ny pt pals me than thee Sind he il hae xpos ee he yi th, hh wld Ty ess frm the Hine kes cnn ai ¥ ose ond se san hc gee el {hh ys del the bhi pref pin ary 3 We will not go through the fll cule OMTIMAL GENE CIKCUHE DESIGN 20 i ert ns it yates ein digs pl aap wteproenseo!§, Fives mga fr pun aie ham serctecaie Reee degradation cate, maxi level, ad stivation hes fo an lerfore be tare by natural selection chest it the (Equation 4.6.5) The delay the dely in the BEL acs to fie ou pulses that are shorter than “(igure 108), ZUR avoids the teition in growth for shor pulses. However the delay papery the Pet ateo has a dsadsontage, because during long pulses, produced onl at dela and ses some ofthe potential beng of the pulse (Figute 10.98). This means thet the ng ‘ome situations in wbich the FEL dacs more harm than good. "io aaess whether the BY, fonfes act svanioge to the eel, relative to sip eeglaton, regres anus ofthe {ull distribution of pulses in the environment “The environment of the cul can be characterized by the probability clssibution of the ration of input pulses, PUD). Let us assume for simply that he pulse ate far apare 4o tha the system tarts eac pulse frm zero initial Z levels (and Y level in the ene af fhe FEL. In this case the average fitness. averaged over anany ipa pulese can be found! by Untegrating the fitness yer pulse over the pute distribution, = fMD) giD) a The esi with higher average nes as a selective advantage hese consierstins map the relation between the selection of these gene circuited {he environment in which they evolve. this is expres! as tations between certain Bes ofthe pulse ditibuion. Hxerisey 107 and {0 show Ital theae relations cow ee solved exactly fr certuin distributions. These solations indicate that the FIL isseecied hy ‘Rane ehvironments and notin others, For example, the FF. is never selected ove simple ‘egulaton in environments with an exponential puise distribution, AD) es On ins ier hand, the FEI, an be selcted in environments witha bimodal pul dstibor Teich bas peubailtyp for short pes hat reduce Benes, nd peabsbity I por {ns benetial puss The oso dey for an F- sch am envionment lea dy {Wat precisely equals the duration ofthe short pas. this delay ites wot the moncoee sft pulses, but hos miniinal negtve impel oe the Htest during lng pala, For {tension ome can dha seit dara that shows Which eit dig ae ‘ther avs Hess (Figure 10.10) this section diageazn shaves thatthe FEL ie owe fitSipe ptinn mt TE -{ 7 GURY 10 Dynamics gen expression and growth nen ash, ade ule aos ong ple lf. The signal § tee tnghout () Sipe regulation wipers roth da ev pes short puis (FF ites the abot ple, thas edad beac ring the og use. he are shes op toa 4 Falasat, (2) Norlin dynamic ea pesion ise on fe oy Tog Yn = i he asf ge relat) aad sproacher seal ste evel 2.0) Noreaied cst (ection in rth ae) deo the pct onl Ct ey ns ate te ly ay 0 ered rth texan eet Fen ection o 25) Net anna gmc tn) ah be na often? GUE 1.40 Section digran for a eminent with oye plac sont pee ha occ ih priya ng ple wk peby Tp. The xi eet of bee open coasters ponte Sw We snp at meter et a than simple relation ina region where bret pulses are common aud the benefit-to-cost ratio of the gene system is not fo0 high. Simple regulation ts superior when brief pulses are rare, When costs exceed benefits, nether cireult is selected, Exercise 10.10 apps this forthe cise of to sugar metabolism systems in E.coli the lactase simply egulated system the arabinose FEL system that was mentioned in Chapter 4.65. 1 hope that this simplified analysis gives a taste for the possibility of studying the selection of gene cixcuits, and their optimal parameters, in temporally. changing 1.5 SUMMARY In this chapter we discussed cost-benefit analysis as «theoretical framework for opis circuit design. We saw that for growing bacteria, the fitness fanetinn corresponds to che cell growth rte, The cost and benefit functions can be directly measired, showing for the lac system a cost that increases nonlinearly with the amount of protein produced. The fitness fanetion, equal to the difference between benefit a cost, has ¢ well-defined optimum in each environmental condition. Optimal protein levels that maximize growth eateare reached ‘apily and precisely by evolutionary selection in contelled evetionary exp ‘We also analyzed the cost and benefit of gene regulation, We sw that gene regulation 4s worth maintaining only in variable environments, In constant environments, regula- tion woul tne 4 be Tos, ais the casein orgasms living as parasites within the rélt- tively constant conditions provided by their hosts ally, we saw that cost-benefit analysis can also be carried out in @ dynamically changing environment, to suggest criteria forthe selection of network motif such as the cohrent FFL, Aecording to this simplified analysis, the FF. can be selected in enviton- ‘ments tht have deleteriows short pulses of induction, which need tobe filtered out by the fiction of the FFL, ‘We cureently have mote information abont the structuee of transcriptional networks than about the precise environment and ecology in which they evelved. One might imagine an inverse problem ~- “inverse ecology” — deducing information about the envitonment ‘based on the observed gene regulation networks This is Based an the ies that optimal ‘routs contain, na sense, at internal model of te environment. Por example, he optimal Adela time ofthe FEL contsins information about the distributions of input pulses, Thus an intriguing goal isto use optimality considerations understand the molecular details ‘of mechanisms based on the environment in which they wore selected, ‘We will continue with these ideas in the next chapter, in which we wil use opteniaa- tion concepts to deduce rules for patterns of gone regultion. TURTLIFR READING. On Fitness and Ewsktion ‘Grow, J and Kimara, M. (970), Ax Jetodaetion to Population Genetics Theory. Mager amd na, SF. an Lona, RE 2008. Groton Experiments with Microorganisms: The Dyas Genetic Bases of Adaptation, Nat, Rey. Gent 487-08 (193. Te Ste or Existence. Davee Proens Gus,Optimality ant Even the Ev Sesto ‘ks, Hail Alo, U. (2005), Optimay and evolationary suing 0 the expression level af 4 prot. Nasu, 48: 588 592 Hast, Cand Dykbhutzen, DA, (1980). The population genetics of Fess cli. Amit Rew Gonet Me 3-68 Selection of he HE Network Mati Deda, Mang, San Al, U 2005). Enviconsnenta slat the dtr ward lop ci ‘ui in yene-egation netwouks. Pye. ily 2 A188 Optimality incites im Msiabosny Heisrich, R. ool Schuster S, (1996). The Regulation of Calla Stems, Khiwer Acadenie abies ares, LU, Fawards jan also, BO, (2002) aserichia ol K-12 undergo apie exe Tun to achieve im seo predicted optimal growth. Natur, 420 186-18 MelendeyHevia, and sor, A (985) 17 251-263 DXERCISES, 10.1, Lining substrate, Proven X ian enzyme that ate ona substrate to provide fitness to the organism, The substrate concentration is L. Calculate the filness function AX, 1) assuing linear vost, € ~ AX. and a benefit that is a Michaelis-Menten term, LoL, 2) = bf. X/0C + 0, appropriate for eases where the substrate. rater thatthe ey me X is limiting. Calculate the optimal enzyme level asa function of Land 10.2, For exereise 101 what isthe exinimal substrate level L required for maintenance of the gene for Xby the wrganism? When is the gene lst” Explain, 10.3, Optimal expression of a subunit 8. Module nits of protein X act together in a multi-uoit complex. The bene isa Lill funtion, BOO = by X*AKe + Xe, anal the cast fanetion ts lisear it X. Wht isthe optimal protein vel? Explain, ', Protein X brings benefit to the cell only when its concentration exceeds X,.s that {X) = OX > X,) gehere O isthe step function, What i the optimal expres son level of 32 104, Cost fanction 8, Derive the cost function in Equation 10.24, based on a iiting resource R, such tha the growth rates equal to F=f, (Ky +R) Each anit of peotein Z reduces R bya small amount « ‘In bacteria cells the resource & often snereases ws the growth rae decreases. Foe cxample the traction of tree ribosomes increases a growth rat slows, because at high geowth rates the ribosomes ase mestly engaged in making new eibosooes ‘his tft cam be add to the model to in sina ost Funeians atthe low to game ofthe pentote phosphate cf. Ther Hil, desi thi inmermediate expression levels 7-clevant tn the exporinantd eon thapton bt nnn veges igh Ase ti = 6 sotto jr al tin eer au “The burden af % prolactin can be describe as a eli ‘sal resource Ry uch that ea ami of pea es he ena FE simount eso that I goes oR = 22. ence, the cox defined asthe tion in geowh ane, asin Equation 10.24 $(2)-f(0), FR UKy# RILAREZVKe RD, ey rte) tis wir the ntti prs of 63) = Kye Rant he Mie = (1 Note tht he ont canner ies base ofthe resmurce Ry thats when Z~ Bone Gin (2) = ad te toc+ 105 to 109 bull sign ici CO 1 hat ent b US, Short ne pes hae a tie eft om gow ne Sr forth nn ani FH ile see lac nel ncn ih vats oY aso ae soar at et tegen tout he ed eX Vue "mea ei pss ah a rcpt ee whan AND it ano is i tne . ann trate hin the presence af hath signals s ecu coral ch lead toa net grow the prod of sy and ate Show tht fe fe fend edt nese On eth re lg He sivatge Satin ‘Toanaye thee ote dependent inpats ln we sth srt the fet af proton of yo AT cos of Zprodastion etal tion growth rate > hen Pn of rotons the ein in grote ZA On theater Bond the vein ofthe Z gone pret caer an ml De calls, This advantage, the Benefits deseo by BZ, te arate BET ag “duet the ation of Z, "The overall elect of 7, un the growth ti and benoit ter pron) p+ bi2) ™ A, ee ha oh Baers heearideiat caesarean | ose‘Now consider a pulse of input signals, in which Ss present at 2 pute of duration D. The growth of ells with simple Lume Dy is given bys ating levels for tion, intgrate over ‘whoo the pase sins, protein Z begins to be produced at rate, aad degraded or Ailoted ext by cll growth at rate. the dynamies of Z concentration are given hy Ue simple dynausieal equation we discussed in Chapter 2 (Equation 2.4.2) aw Mo peai, crm0.3) resulting in the fasilar exponential approach to steadystate 203=2,(1-¢ 0.4) For ang pulses (De >> 1) the protein concentration Z.is saturate atite steady state value Z~ Z,, Protein Z has net positive efet om cell growth (0) = np +b¢7,a0>0 (95) provided thatthe benefit of exceeds its prodction costs de> ng (P09, Short puises, however, ca havea deleterious effect on growth, To se this, emer short pulses such that a << 1. During the short pulse, the concentration of rises linear with time (as we saw in Equation 24.7), with a slope equal io the produc tion ste 0-61 107) Since Z cannot reach high levels during the short induction uae, we cam wse ¢ series expansion of the benefit function b(Z) ~ b' 2, where b= d bid Z. at 7 Using this in Equation P10.2, we fi thatthe integrated growth rates a quadratic Function ofthe duration ofthe pulse, D (plotted in Figure 10.8) 0) = Fen eb b , apo bp eros) Important, the expression of % eauses a reduetion in grovel f(D) <0) for pulses shorter thaw a etical pase duration, D,, fal by salving @ (P.) = 0(Figuce 10.8) b= 20 eri09) oy Pulses with 9 = Dare atthe break-even point, because the east exacty equa the benefit, Only pulses longer than D, give a net enefit to te els. Thus sole eR Tation leads to reduction of growth in environments that bave mainly shot pul eve though Z. confers « net advantage for suficiently long, input pes (Figost 10099) 10.6. Conditions for selection of PL. ove sriple regulation, Exercise 10.5 showed expression of Z in response to short input pulses sedsces fines, Hence & cel that cam avoid responses to short pulses, and allow responses only to persistence pulses, can be advantageous, As we saw ia Chapter, the coherent FH. can por this typeof filtering task. fn the coherent FFL, 7 is expressed ony at dy ow afer the signals appess. Thus, only pulses of input signals longer than the dehy Sneath FH, wld a exe, However the fering ost puke lisnlvantage, because during long pulses, Z is produced only at a dele and sis Some athe potential beet of he ule Figo 0). ase ter the FL confers net advantage to the cells, elative to simple regoltion,requies 2st ofthe dstebuton of pulses inthe environmen. The environment of he cll aye chacacterized by the probsbilty distbution of the duration of sap pies PO ‘Assume thatthe pulses ate far apart, so thatthe system starts each pulse fem 280 Siti] 7 levels (and ¥ levels sn the case of the FFL). In this case, the overall tne averaged over many cell generations, can be found by itegrating the Fines PEF pulse over the pulse distribution, Find conditions for the seletion ofthe FFL over Sonple regulation, Solution or simple-egulation circuits, the integrated fitaess includes an integra over 3 possible ses, tines the fitness per pulse @ (D) = J eeoan oo) i than Tog Fest For FFL zeit, prodoction starts afer a delay Ty. Pulses shorter thar Tou inno Z production and g{D < To) = 0. Lang pies begin to be utilized only af the delay Tyg 50 that their duration ie effectively D ~ Tuy (Figure 10.90) resutio® ina contribution inthe integral only from pulses longer than Tos! regulation ease, Equation P10, Is equivalent 1 an FFL Wh Note that the simpl Toy =.BAZ om CHAPTER |) contin for selection of BEL over sil aed fies exces that of simple eects and is positive: Wo, > rage Grd era) Sienple relation is selected wht its integrate fitness excecus that of the FE as? Pre oe? (wis Neither cireuit is selected ther wise (Bj, <0 ad Paya € Ob For the purpose of this comparison, the BFL is chosen to have de apriaal value foro, that maxi ies yy), bocase natural selection ean tue this parameter to best adapt tothe 107. The PPL is ot selecid in the case of exponential pulse distsibutions. Analyze the average ness ofthe FF and simple egulation in a environmen in which pulses thowea conatant probability per unit tise fo end. So neetial pulse distribution: ‘environments have an expo- v)= Dye" (Pio Soletion: Using Equations P10.10 and PIU.L, we find tbat Byc2 J ope (=a MaD=e" foge"™ DaD=e Oe Dee (Pins) has, the PH. is over slated since yyy > Wa, seth probability 1 = p- Analyze the conditions for selection of EFL, and simple regulation as. Furic ‘of pad the ratio af the bene io of protein 7. (Figure 10.10). Solution: the short pulses D, are nonberelicial, since they are shorter thas the exticl pase width at which pone Z roaches the break-even point, D, <1 {Hgure M18) tn co trast the long pulses Dave henefcial and have a benef af approximately applying Exqstion 0405 AP, +b07,1D, 0 crt) In this case, itis easy to calla the optimal delay i the PP: the eptienal delay is Ty Dy That the optimal FHL has a delay that blocks the short pulses precisely longer delay would not further filter out short pulses, and would only reduce the ‘vena of the kg pulses. The condition fr scleeton of FFL over simple regulation, Found by soving Eaaitions P10.10 and P10. 10 fd Dag = (0 ~ BUDE) ~ HB) Dy = poBD, and yx = {L ~ pO) ~ 8) (D, ~ Dy). This shows that the Ls move fit when the prabablityof short pulses excceds factor elated to the rato of costo beni of 2 nb pote P40.) per c ‘the phase diagram for selection i shown in Figute 10.10, When the ratio of benefit 0 cost 92 is srl, either cicult selected (costs outweigh benefits) AL Jarge raative beats the FFL is selected if short pulses are common enough ~ thal Is fp is lange enough (Equation PI0.17) I short pulses are ear, simple regula tion selected, Ata given p the higher the bene sslection of sinple-regulation circuits. 109, Why i FP seccted in the ara systom but Hot inthe le spstom af E coli ln tis ‘exercie, we wil apply in a qualitative way, the results of execise 10 tothe case ‘of sugae systems in cal, Why isthe FPL selected in some supar systems, such as arabinose wi ion (ara syste discussed in Section 46.5), where snp regu iow in slectd Solution otlers, sch asthe lactose system (ae sjsten}t “The magels are only simplified toy models, but let us proceed for demonstra: sion psrposs, Both ara and lar systems share the same transcription activator, X= CRD, simulated by S,= cAMP, a signaling malecue produced by the cell upon gluccue starvation, Thus, both ara and lac systems have the same S, ube distribution, However, these systems dif in the benefit they yield per sugar molecule the benefit-cost rato, ,)B, appears to be dtferent for thet syste, "th benefit per latoxe molecu, hie is grater than the benefit per arabinose mole spit into glucose + galactose, le (approximately 70 ATs per Tnctose molecule vs, approximately 30 AYP’ per arabinose molecu. tn ation2h CHAM a ro... vo to its smaller benefit, the enst of the wa system miay be fr the fac system, because there are at east seven highly expressed aa proteins the metabolic enzymes /rsB, AraAvand AtaD. and the pumps AraRand AraEGH), compared to only three highly expressed lac proteins (Lac7, la, and Lach) “Thus. the parameter NZ.) for the ara system miay be more te the lef in Fi ‘ore 1010 eatve tothe fae system, fvering selection of PF, in the former Ihe delay ithe PP. can be tunel by natural selection. As mentioned in Chapter 4, the delay inthe au system appears to be on the limescale ofthe deleterious shoet pulses inthe environment Cascades vs, EFT Repeat the calculations af Fxerises 1h and 107 for 9 eas: cade X-> ¥ > 7, Show that cascades are never niere optimal thaa PELs for envi= ronsients with pulses of inp signals. Explain tie ces (Advanced students) The cost and benefit of SIM. X controls gees Z, and 2 in 2 single-input module (SIM). Gene products Z, and 2, assemble nto complex, such that a, units of protein Z, fst assemble into subunit Sand then n unis of prot 2, join subunit, and form the final proact SX hens to be produced strate at time =0. What are the optimal activation tareshods and production "ates for genes Z, and 7:2 Use logie input fanetions. The production costs for Z, and Z, are, and and benefit only occurs when a unit of, is aoduced. owarer 11 Demand Rules for Gene Regulation HT INIRODUCHION ee The control of gene expression involves complex mechanisms that show Torge variation in sign. In this chapter, we will discuss whether the mechanist thats used in each eases 4 resalt of random historical choige, or whether there are rules that can help us to under stan the design in cach ase, For this puspose, we will return to transcription networks, and attempt to deduce rules for gene regulation. The specifi question we wil ask i: why are there positive and negative mades of regulation? ‘Thats, why are some genes regulated bya repressor, and others by an activator? What determines the mode ofthe regulation in cach case? [eis important to first note that activators and repressors can achieve exactly the same regulatory goal. For example, a gene thats fully expressed only inthe presence of sig ral (Figure 11.1, can be regubsted by one of eo mechanisms: Either an activator binds the promoter to ativate the gene, oF aceprssor fll off the prammoter to activate the gene “These 1wo mechanisms realize the same input-output eelationship: Expression is torned ‘on by the binding of an activator in Uh positive mode of control and by the unbinding of ‘repressor in the negative mode of control. More generally, gene controlled by N regu tors, each of which ean be eller an activator ora represor, has 2 possible mechanisms ‘that can generate a given input-output mapping. "Among these equivalent mechanisms, evolutlnary selection chooses one for each sy8- tem, Are there rules that govern this selection? One possibility is that evolution chooses randomly between equivalent designs. Hence the selected mechanism is determined by historical precedent. Another possibilty is that general prineples exist, which goveen the choice of mechanism in cach system. “The question of rales for gene eegulation was raised by M.A, Sovageat in his pioneer- scriptional control (Savageau 1974, 1977, 1983). Savageau found that the aeyl” “ x” NGAI 111 Tanaagtion etn sia fe Pi contd Gt a heat sag lap te 1-2 tc bry, sata Ari. Incase ohccopena slowness. hay tose ahh cet peice esse fh Nga at pre. Wh he er sgl Se eth ae S260 oboanlty epee he casrthe gm ep ale ze When ther pest {Fei ots ohh eas om oe ay ssa (7 ) hers pe as an Sas anloajaic? sahsesd pie court 01 canapunise necoloanea seen Neqoing tentang Saran an sot ofp tole of control is correlated wit the demand cleined as the friction of tine i the nate tal environment that the gene product needed near the high en ofits regul regulatory range, High-doniand genes, in which the yene proc steve most ofthe tise, vi positive (activator) ctl, Love demand nos of the ten to abe proaict isnot required ve, bond to have negative (repressor) contol This demand rule appeses to bein agrecment with over 100 gone systems (Savage 1988) fro B,colé and aber arg sms, where the mode of conte is known andl the demand can be evaluated. Ia this chapter, we wil describe demaay rules fae gene regulation base on isle di ferences between the modes of contol, These differences are due tothe fact that biologi tal intermalstte ase prone to error, which lead 10 evors inthe output The errors ees io a reduction of the organism's fines. ‘hs reduction i called the error-toad. Equiv lent mechanisms, which implement the stme input-output gelationsip, can differ in their ‘error Laas Afior describing the Saeagesu demand rules, we will examine the proposal dat evo Tution selects the mechanises that minimizes the error-toadl, We wil sce hos error Joa wanna nea explain the eounection Between the mode uf regulation andthe Aleman fora gone inthe organisons easinosments rarely need lated by repressors and genes commonly nocd at fll expression tena to be regulated by acivatrs, This heory can be extended 1 the case of multiple raguators. We will ee hoe ereorsload mininiation can explain detailed Geatures of the seuetre of the EE colt Jae systems, We will also discuss he ceteris for when selection acco to these rules dominates ower ‘Move generally, the goal ofthis chapter (and or encourage the point f view that rules canbe sought to wnderstacd the detailed suctare of biological syste J oF Ue wan goals of is Fok) isto 2.2. TIE SAVAGEAU DEMAND RULE M.A. Savayeau nore strong correlation between he nae of bacterial gene regulation te probity tht the gene is Fully expressed a the environment." Firma 1s wage defined the demand fora gene system 38 fll “When a system operates close the high end of ts egulatble rage most of the time in its natural enviconment iis sid to bea high-demand system, When i operates atthe 1 i nalural envionment it ssa to be Jove end ofits rogulatable ange most of thet alow-demand system” DDewsand corresponds 10 the frequency at which the funetion carried oat by the gene system is needed within the ecology ofthe organist, For example, system that degrades certain sugar for use as an energy sont is in lowe demand i the sugar is rare in the ‘environment. the system is in high demand a the gar is often swale, A systens that synthesizes an amino-acid iin low demand if that amino-acid is commonly available in significant amounts in the enviconanent ~ demand islow because de-nove synthesis ofthe amino-acid i not lien needed. Conversely 2 system that synthesizes aa arsino-acid that 'sonly rarely availible from the ouside i in high demand Bac of thee stern can be regulated citer by a pressor or by an aotivator that is, ‘ther by a negative or by a postive regulation mode. Ihe damund rule may be stated as follows. “The molecular mode of gene regulation is correlated with the demand for {gene expression inthe onganisn’s natural envieonmient. The made is positive wher ‘he dco is high aad negative whew the deman i low." Thus, early needed genes tex tobe regulated by repressors, commonly aeeled genes by activators 11.21 Eviconee fa the Deana Rule inf, col fo test the demand rule, one aces to have knowledge ofthe mode af regulation aod of the demand fort gene system in question, For this purpose, Savage collected data on Use natural enviconment ofthe bacterivt» coll. One ofthe principle habilas of Ecol isthe imestinal syste of its marnmatizn host Studies of this environment suggest that llfreat sugars art aevino-acids have diferent abundances, Some sugars are taken up readily by the bogs. and see thus ravely available forthe bacteria. Other sugars are lest readily absorbed and ate mach more consmen in the bacteria environment. This leds to ‘he following estiated ranking of sugar abundances: -plicose < Degalactose < gyceral < Dexylose < Leyljcose < Lemunnase < Lficose < Torhamnose < f-arabinoseJ 11 Moteur Mad oF Reuitnn ad Depa Ge Dap Ge Sen ihe emt Degradation tom “Mode tveguaion —Tageltor Tewand for vcd in preteen) expres Frain Tone Tae igh Face Posie ack i Gate agave alkcas Low yer Nogave pe toe actos Neate 1a Low pie oie cat ah Matase Poative Murr gh tapos ost as sgh Nye ote conn igs Proline pean) Nagaine vk Law Pettey) Naptne put to (Savageau 1976, 1977, 1983). The sar Ietose is also care sugar inthe environment of a because tis cleaved by specialized enzymes the upper intestinal race Silty est ‘mates for amino-acid abundances ar: glycine >leacine> phenylalanine > hstilne> alanine > serine> valine > aspartate» prone > threonine > eytine > leucine > methionine. A comparison of the mode of control of inducible systems that degeate nutrient is showa in Table 1.1. For example, the sugar galactose i sldom present at high concent ons in the environment of F cli, which coezesponds to fow demand fo the galactose tenes that degrade and utilize this sugar. According tothe demand rule, the galactose sys ‘em shoud have negative contol. This isin agreement with the repressors Galle and Gals ‘that control this system. On the other hand, arabinose is found at high concentrations, «comesponiig to high demand of the arabinose wtlization system. ls mods of regulon is positive, withthe activator AraC, in agreement with the demand rule Simla results are shown in Table 11.2 fora qumnber of biosynthesis systems that pe: ‘dace & compound in the cell. The expression of these systems i reduced if he compotnd that they synthesize is available from the outside. For example the arginine biosynte Jol 1 Mine Reliant Dl Rint Cen See ‘onatbae stem Induced Mole of elation Regular Danna for Inthe abner of satan roreion theatres ofonintanes) sane “ane rome Tak nine Poe cya Ilene Poste Ir tecive Poste ep v0 sine Baste yt *iytepan epi io yen eine yt ace ela sis systom of F coi produces the smino-acid arginine that is relatively abun i the natural envirooment of the eels. The corresponding biossabesi sytem is thus in low ‘dematn, nl the demand rules allow one to predict a negative mode of cote. Inbed, ‘his system is regulated by a repressor ArgR, On the other han, cysteine biosynthesis, 2 Ih domane system due tothe low abundance of cystine, regulated in a postin msde by the ctivator Cys, “the cule also successfully predicts that systems with biosynthesis and degradation of a compound, tend to have opposite modes of regula tion, In contcas, systems with aligned Junctions, such as cransport and wiiliation of 2 ‘compound, tend to have the same regulation move (Savageau, 1977, 1989). These predic ic oystents tee Ive sponte agonistic fasetions, such as tions flloe from the semana ale bes deans, and systems with aligned functions tend to ave the same demand (both igh ‘oF both tow}. Note that predictions ofthis kind do nat require kewedge of the precise demand forthe systems ‘More complete data on gene regulation networks tends to support the demand tule, ‘but some exceptions tothe rule ae also found. One possible example i the biosynthesis system of fysine, an abundant amino acid. Since lysine is relatively abundant, the demand {or its de-nove synthesis is lo. However, this system is controlled in a positive mode by the activator Lyi in contrast to the predicted neyative mode, ‘The definition of demand is offen tentative, because we lack information on the ecology of the cells for many y= tems. On the whole, however, the Savageau demand rule seems to capture the mode of ‘many of the known gene regulation interactions in bacteréa where the demand can be reasonably estimated, 11.2.2 Mutational Pxplanation of the Demand Rule ‘The demand rate was deduced by Savageau based on the effects that mutations have the two modes of regulation, This theory first assumes that there are ao inherent fen: ‘ional differences between the two modes of regulation, Thats, precisely the same modu Iition of gene expression in response to a signal ean be achieved ether by a repressor Diruling the promoter or by an activator unbinding from the promotes. ‘This assumption suggests that one should focus on the behavior of nants that are altered i tke regs {ory mechanism ‘The theory next uses the fact that mast mutations im highly evolved structures are det imental, and very few mutations are bencicial, Consequently, most mations i a rep- latory mechanism lead to Jos of regulation. In the ease of positive mode loss of regulation results in super-repressed low expression, because the activator is no longer functional In the case of» negative mod, loss of regulation results in constitutive high expression byes the repress i no futivnal Thus, mechaniss with different maces respond In opposite ways to mutations, Tie result of these considerations is thatthe two svodes will fate iferenty in a given environment (Table 11.3). "The positive aiode of regulation is avore stable against[API 1.) Scton a Matas oe Dn Mader of Ca Aig the Maan S wae Tega Hh Regulation seks etn how Rega Ragen stad 1utations ina high demand environme demand exvizanment and the negative mod is mre stable i los ounderstand this, considera positively regulate geo helena environmen the wik-type organism will induce the gene high level most ofthe sine. Mutants wis have tost the regulation, will not expres te gene Asa resi they will beat disadvan tage mast ofthe tine, an will be fst from the population. {tn a low demand environment, however, expression of the gene will be shutoff mast ‘af the tint, The tutats, wo have lost lke activator, will he unable to express the gene These will be refatively weak selection ayains this loss of ulation in this system, because the yee is rarely needed at high expression. Super-represed mutts will ccumlate as «sul of mutations, and the functional regulatory systems wil be lst. Tense, a positive mou is moe sable in high demand envizonment than in a low demand environment the opposite when one considers a negative mode of regal: ‘mutants will be stoongly selected against In a low detsand environ sent cause these rutaets have los the repressor and have un-needed high expression ‘The high expression fas a fitness cost and lead to Jos ofthe mutants fron the popula tion, In contrast, there will be a relatively lower selection presse against mats in a high-cemanl environment, because the gene needed at high levels most ofthe time. Thus, according fo this argument, mutants will accumu over tit, and the negative regulatory system will be los 1 summary, the mutant selection theary suggests that negative mode is stable i low ‘demand envizunmens, and postive mode fs stable in high demand enviconments [1.25 The Problem with Malan? Seder ion Argun Mutintcecton arguments are valid ees if there isn intestines auhontage to one of the to mois of ented. I mich intron ditterensey exist they would daa Ove he ferential effets of mutations. The fitter mechanism would rely take over the popu tion, tn otber words, mutational ects ace second-order with respect to inherent diflerences in the wild-type mechanisms. fn the next section, we will develop a theory to understand the demand rules based on intrinsic ferences betwoen the modes of regulation. "These dif ferences between modes of regulation correspond to thee resistance to erzors, INIMAL ERROR LOAD. es based om inherent ftness tal, 2006), The idea i simple 16 Ht RULES FOR GENE REGULATION BASED ON We will now describe a framework for deducing dem 1 ltfrences between the Ive modes of entra (Sh srt: The mal ssn faa san eal syste DNA Se he ‘oounel tightly tm their veulaory protein are mare protected tomy erro a fies sites, hs is because few sites are expose 10 non-spectic binding ‘ese ing CEOs Jead to changes in gee expresso, shih esc the uganisn ness This esto the evalua errno shasta so tose mnie th thcbNa rotated moe tetane sn ey peel mH hdc thdonaedr nn ie goe eg y8 sctpim ate whan seat aren ure) i 8 se Trae no vp: bi i,m st sea wk Be nies ee el re ny std uo and brane pot Whe hese ony sees ne loa Tetouan the oxen lo tt 8 thc The ae atin one ca he eo tas seem a fee DNA sete ea eon srt ates gy Boe 8 thereto rc rset [he eat snes err conned ith fre atthe rt ee ers conta th he oe tasters heen Ce wag eof torneo the te (eae et aly 202, Senge en 26.1 ‘Sao pss beaten a ey he eer ‘Tinea changin apna oasis, ding a nog vents and neyitive coli Sowedemsand environments in oth cs site i foe, Us i the Klealized patie, 10 lesiquated regulator the ste Is protected fr these factors i cote et This non-specific binding can ta rection in the Hess of e kam act to reduce or ineremeexpression, ciosttencting ans tothe site, serosal _ Tending to errs. A second soiree of ertor arises Feom residual binding ofthe designate any eases, the afinty of the inactive (ante eg eel the pin wow the eg ca am 1average rection in fitness fora repressor, taking into account only cers Grom the free Se, is thereore Fy Da, aun ‘Hur an activator, the fie site corresponds to low expression, which occurs rsction [:pof the time (the fraction of time thatthe genes no in demand). Error in the expres- sim level Tea to a elative fitness redaction of Af where the subscript 0" denotes the love expression state The average reduction in fitness For an activator, taking int account ‘only error eon the fr sie, is therefore By pak ay In this simplest case, a represcor will have «fitness advan alent activator when ithas a lower ertorload we over an oberwise equiv- “ty 3) Using Fquation 11.1 and Kguation 11.2 in Equation 11.3, we see that rep advantageous when the demand is lower than a threshold determined by the ratio of the relative reductions in fitness Pte afuagy us) Thus, repressors are advantageous for low-demand genes, and ativitors for high sdemarid genes (Figure 11,2). The reason for this is that repressors in lowsdemandl genes ‘and activators in high-demand genes ensure thatthe site is bound to its designated regu lator mos of the time. The demand rue therefore minimizes the fraction of time that the site is expmed to errors. He HH SITICTION PRESSURE FOR OPTIMAL REGULATION, ‘Con etrorloal create a selection pressure slicient to ease a relator system to be Conder a wild-ype population with a regulatory system in place. Suppose that conditions vary, lading lo a permanent change inthe demand forthe gen, so thatthe epposite made of canal besomnes optimal Mitants with the opposite mode of eonteol arise i the population, by genomie mutation fr lateral gene transfer from other organisms!. These mutants have aloe ereor toad, and hence a relative fitness advantage, which is equal to By» By in the eae of a repres: sor andl Fy» Fy in the ease af an activator. "The mutants can become fixed if their relative replaced by a system withthe opposite mode of cant? ‘Fite re wd carci exam ert sme tpry in amas ihr a pes oh xtester depo the pan a eo we rap ea Ve 3 Cay To; Nomae et a 99) Ore ce eke me msg ah lange sa ae ina cw at Mh aaa 19 Coe TPE Feet aan? 3 FIGURE 1? Stet digi fo oad risimiratio, Each ego csp the made ‘ed withthe sae eet The verte ane th rand pnd a heft fe the pen proc ved at alexpein Thee ans een a thins eos ing eo rosin es the plied eect ana finess advantage exceeds. a minimal selection threshold, yg: The selection threshold Say has been estimated in bacteria and yeast to be in the range JO%-10° (Hart! eta, 199%, Wagner, 20050), “The condition for fixation ofa repressor mutant is rus Fg ~ By > Suns whereas the condition for Sxation of an activator mutant Is Ey = Ey > sue These inequalities lea to a selection diagram (Figuee (13), in which the error minimizing regulatory mechanism becomes fixed ata given demand p only if the ratio Sof (f+ Af) society smal JIncases where the fitness advantage fs smaller than ¢, there exes a gion in parameter space where histrial precedent determines the mode of conte One cam estimate whether the fitness reductions caused by expression errors, Af and Af lead to selectable ercor-load differences. "The fitness as a function of protein expres ‘sion was described in the previous chapter forthe lac systema of E cl. Te fitness func ton indicates that a 1% error in expression leads to relative fitness reductions Af, and Af, ‘on the order af 10°, which is our axders of magnitude higher than the selection thresh ‘ld Sy Similarly, Wagner estimated that in yeast, a 286 change inthe expression level of ny protein is suficient to cause fitness dilferences that exceed the selection threshold (Wgner, 2005a). These considerations suggest that even minvte expression errors end to cerror-londelfets that can dominate over historical precedent in determining the choice ‘of regulatory system, 11.5 DEMAND RULES FOR MULT M So for, we have analyzed the demand rules for 2 gene with a singe regulator. We have ‘seen that activators ate better than repressors for segulatng high demand genes and that repressors are better than activators for regulating low demand genes. In both cases, the DNA site is bound for most ofthe time, minimizing errors. Let us now turn to systems ‘with multiple regulators. For clarity, we will consider in detall she Ja system of Eco ‘even though the present considerations can be generally applied to other systemana CAVITE UY 8 for 1 wal OH OD mode fon in he fi of ening mh GLE IL Skt an eeuriiniing ea tnt ei 0 ethan marke acoso pres 8 e5 ve Seer cn can ce Fd et tye olan she en an ten aed tera seen tas while mb rene sma ak een whan sting. nt oe aa haste minnaladerton aanns ped ston, ete hes AA ne a of nanan fates ae afar at sh pte ae 0 sons 1, the Lac proteins transport the sugar lactose isto [As mentioned in previous chapters the hae p he sai te Pion The sem ht i see pum tie pty cd ponte BSE: @ikShpctapeumr nuttin he pecs fe wi 2s two 1 sugars onto the expression levels of the fac proteins, is shown itt Figt ae tpn ny plemented ay wo ge he cL Marae hes ans co hc espe it i Fahim xp the tac Wh hase ens Sis mss oot navi hitb eproson in he eens cd dace hen os pei te sO aoe fe gacetng ection oh le sys a “fe canto etait te DNA ings sn Recpee luc lac sytonn is showen in Figure Hb, There are four possibe bincing,state vei see Ae Sarat at seared ees ints dete Canyon ya ashe 1 oe oC {tw gs Lol (et ey ay a bn aa eye of pe the fel hen ve aco HMA ROLES FIR GIST RIGKEALON 20) the envionment. Axa result the binding state [Naf des not eorzespond to any fpete Sate and may therfore be called un exeluded state, lis exprosinn level wis experi ‘mentally determi by asin artical inducers sch ay CPTC, whic ae not subject ty inducer exetusion, The matozlty occuring mechanism, witha ghacose-responsiveaetivato ad ete Fesponsive xpress only one uf the four possible mechanisms in whieh thet eeu lator can have ett mode of cova jure 1.8). The for mechanisms ca he denoted RK, RA. AR and 4A where the firs letter denotes the nies of the glacese relator the second leter denotes the mode of the lactose regulator an the designation A/R ere spon o ativatorirepressor The wild-type la system has the AR wich ‘ator CHRP and repressor Lal (Ligure 1.53, with sti “these foe mectnnisai al rp the inpot states nthe express levels inthe san ‘oy The mechanisms difer only in the promoter inding-states thal vorvespotil (oe cach input and ourput-tate All four mechanisms have ilucer exclusion, ane! thas have ay excluded state athe he identity ofthe excluded state dsfers between the mechaisnis (Pigute 1.9 The eccued state is [ERPLaet}(0.0], 0), [L., flall inthe AR, AA, te anal RA mechanisms eespoctvely. {tus now consider the cero in this system. As before, feos are assumed to be denote & population average Tints use a Tay na us. DEMAND RULES FOR & RLGUIATION © 201 48) Compute the mean reduction in finess due to variations inn the fully nde sate the average value of Z in the population is 7). Answer: A--0.1% Demand rues for developrwntl genes: Consider a cell which, during the develop "ental proses f the srganism, an assume either fate A or fte B.A sel of genes Cy 's expressed in fate A and not in fate B, and a diffe sot Gy is expressed in fate unl not fA. ‘Uhiscell-fae deision is regulated by two transceiption-faetors X and ¥. X activates its own transcription and represses the (ranscription of ¥, whereas Y ‘ctvates is on transcription and represses the transcription of X. Barthermore, ‘ranseriptionlly sotivates Gand sepeesses Gyan Y has the opposite effect, acti valing Ga and represing Gy (0) Draw the iransription network inthis ease (b) Pxplain the mode of egulation ofeach get in tems ofthe demand ule Error load of two-input genes: Compute the error-lats ofall posible four regla- ‘ory mechanisns for the lac system, accortng to Table 114. Compute the condi tions (the range of Pye and fy) én which each ofthe four mechanisms Is optima Compare your results to Fig 116, Erorload ofa feed-forward loop (1) Consider @type-t coherent FEL (Chapter 4). tm this gene circuit, activators X and Y activate gene Z, and X also activates Yo that steady state, ¥ levels are zero unless X ig transcriptionally active. The regula. tors X and ¥ respond to input signals 8, and §, the promoter of gene Z is activated 4 am additive fashion by X and Y, such that st steady-state the expression level 1s Zea XbYs {a) Plot the steady-state eelationship between input states ( [ee.¥4] and output states (7%, (Similar to Fg 11.4). internal states % 2) forall four combinations of Sy S, =0 ot () Isthere an excluded stote inthis case (a steady-state)? (©) Repeat this for an incoherent type-l EF. in which the oetpat ix Zoa Xb Y “Assume that bs, and that at steady-state, ¥ levels are zera unless Kis transcrip tionally ativecuanren 12 arte TE Epilogue: Simplicity in Biology ee 1 hve ano inserting this book ad fave gone over many dats 1.8 DAP sll have a sense of wonder whe feadng the chaps. The wondee comes Dees 7 svorks of thousands of ineractng components are generally incrnrehenible, TH out prin yeas tat immensely complex blogal yen woul be understands that despite te fact that biological networks evlied to function and ot 1 be CNP oni sniping pills canbe found hat mak lois ign dette woos “Ths epilogue wil discuss simplicity in several aspocts of biological networks. We vill seve spy sacar al times inthe abit ofr mpd moo fegolatory networks i in sevursing design principe ‘One lel of spicy cure in the structure of olga networks chon ate huge einer of posible interaction pastors The arp ot Bole! snort bulla goo approstation fou onl ew ype rcurting ies Paltens called network mots Each ofthe network motfcan perform deine information proses ivan network mois found in sensory transcription networks a thelr Funct Summarized ia Figure 815. Fr example, feed-forward nos can act 880-50 ter pe erates an ep aezlss, Naive ue 2 PEL ‘elie responses Singleinpi modules can geste temp prograns of "eS Rae ame mens bdo How = cvs beces mee compl “The same network matifsappeat In the sensory tanseriptm netnosks of dere ngnisms ei importan ostess tat the sislariy i circ patterns does 01 FEE sarily tem from eect duplication, Hvolation appears to have conserged on (hs SPN betwore motifs agin and again in diferent systems, suggesting at HEY H8 A Seu ofthe fection Tes actions can berated experimental 4 syste ‘Network motirare embed inthe newark and are connected to each othe PP tantly the usaf appear to be ened na way te allows ee 4 eye ~ large newer notions. The ive eTBate CHAPITR Ee their fuetions even inthe presence uP adaliional intersetions, This property i eae 40 the particular way that the motifs se wired tngether. In many systems, retwork mots appear to he connected to each other in ways that donot spoil the independent Fanti ork, eat partially based on the Ionetions of sniviial motifs. Snape examples inclde the way that thyeenade FFL are ‘connected ln cach other to form a malti-outpot EFL, "This ptlern preserves the fection: ality of exch three-node FF. (sige-scusitive illeving, ete). In akiton, the multi-output FEEL can generate rater elaborate programs of expression fing between ont genes, 28 We saw in Chapter 5. Asa result of tue way the miaifs are embedded int the network, they eu tliat in anany eases, be eansiderd as elementary circuit lentes, ple ways which mutifsice wired together, matfscan act as le rmoutary xreust elements due to the separation of tanescales oF diferent sneractions. the Strang separation of timescales hetwern different biological proceses i a general princk ality of each motif allowing us to understand! the ple that is found in virtually all of the networks inthe cell. Ht allones us to gers the dynamics on the slow timescale by using steady-state approximations forthe interactions fm fast mescles. or example, teanseriptional motifs that carry ou thls computations fa slow timescale of minutes # haves can be understood, at least schematically. as if they acted in isolation, despite the fact shat they are embeded in additional feedback Joop on te level of protcin-protein smteractions that function othe timescale of ec fonds. In short, biological actwurks can he understond, oa first approximation, i terms ofa rather limited sot of recurring circuit patorns, each carrying ool conpatations on a allerent timescale Tn audition to the reuse of network motifs, bislogied networks haven additional Important structural feature: modularity (LJartwel eta, 1999; Ihmels et L, 2002; Segal etal, 200; Wolf anh Arkin, 200%; Schlosser and Wagnee, 2004). Mest bollogical func Lions afe carried out hy specific groups of genes and proteins, so that one ean separate the structure into funetional siadles, For example, peuleins workin enregalated groups such as pathways and complexes. Transcription networks age neatly decemposable into Singleinpat modes and aul input dense overlapping eyions (DORS, a8 described fa Chapter 5, Signal tramlaction networks display distinct signaling pathways shape! as ron modes, discussed in Chapter 6 ee. Ihe muds in biological snetiorks cat be compa on the metaphorical level to the ynodules wd in engineering, such as sulvourines in software and replaceable parts in machines ‘A working definition of « mde is set of notes that have strong interactions and a function. A module has delined input apd outpot nodes that control the snter action withthe est of the network. A modale als as internal nodes that do not signifi cantly interact with the nodes outside the module. Modules in engineering. and probably also in biolngy have special featnees that make them easily enfoeded in simast any’ sys ‘xia meals shout have ow tsypelance, so that adding on additional losenstgnen client sho not drain the eupat te esting elie opto some Lin). ‘Why does mualarity exis npc tone that ‘evolved networks ate modula. he opywsite is trust own solations ae ape te. Fae ex in biological networks? Is ie simple compastersiemulaions of volutes Iv evotinnary sisal sion of WILOGUT: SIMPLICITY IN BIOLOGY e235 2 Fed goal evolution > rron sane gi cay sey oem % Md bg “2 : ont ¢ Modulany varying e Mid i q aaa gy ae of me _ stig es (and ptt computes NOTANED Tector oes oe ea ‘Geyer 2 generations Noe tha Gan G share heme sabrobems (enna 8 NOM tee a, -modularty varying goals towand G, and G.. (l) Madulae structure of the networks evsved ialer modular ‘etworks i evolved by randomly adding, removing, and changing connections betwee odes = and ever duplicating and recombining parts of the networks ~ unt the net works perform a given computation goal, thai, until the networks give the correct ont Putto-npat signals, Unlike iological networks, simulated networks evoled in this way are usually nonmodulae (Figure 12.1, They typically have a highly interconnected struc ‘ore that cannot be decomposed into nearly independent subsystems (Thame, 1998), ‘Viewed in this perspective, the modularity of biaogical networks is puzaing The evo- Itionary simulations make it elear that modular structre i usually less opie than {ally wired, oonmodular structures. Afterall modules greatly limit the number of pos: sible connections inthe network, and usually a connection can be added that reduces mdularty and increases the fitness ofthe netwoe, This isthe eeason shat the elation: ary simulations almost always display a nonmodulsr solution, [A clue tn the season why modules evolve in biology cam be found in engineer Ing. Modules sn engineering, convey an advantage iin situations where the designspecications change fi sted works git fer a ircto tine, New devices orsltwarec radules (Lipson et, 2002) Silly, i teal enviconments that change over tine (G Kirschner, 1997) Indeed, modular networks can, in some cases, evolve i simalatons ia wiih the evolutionary goal changes over time. Importanty, in order for modularity to arise spoutancounly, the gouls need to change such that each nev goal shares the same ‘sulyproblens with the previous goals: Fach goal is composed ofthe sume sot of subpro Jems ina liforent combination {Kasbtan and Alon, 2005). Unuler such modular varying, goals, networks raplly evolve chat have high fitness to the cuveun! goal and every time the goal chang, enpidly rewire to satisfy Une new gos These networks are highly most lae in strvcture (Figune 12.1b and). They include a modute for each of the subproblems shaved by Ue goal. Iisa though he network learns the shared subpeublens ane! crates ‘specific module for each subproblem. Hvery tine the goa changes, these modules noed ‘only be rewired in order to satisy tbe new goat If he goal stops changing fora sulicent Jengthof time, the networks inthe similation begin te lase madularity ani eee toward 8 nommodalar desiga, a design that is typically more optimal (eg, uses fewer compo- nent) Uhus the ability to recontigute and adapt to new conultions may be one force that helps toa oe ely constructed adult biologic net from ensting, wel svt and Jntaia modular structore io biological systems. Inadition to the stuctural simplicity associated with modularity nd the small num ber of network miotfs that make up the networks, there Isa second level of simplicity. This simplicity Is fund in the realm of models f biological interactions, in the ability lo treat regulatory circuits with simplified mathematical models tht eapture the essence ‘of the behavior an have a eeriain degree of universality. This dbstact mode of descrip tion is surprising because it contrasts with the complex and idiosyncratic biochemical rnvechanisis by which each protein cares out its function. ‘hese biochemical particulars are astoundingly rich, Barogue, but an the level of the dynamics of transcription circuit, we saw that one can use rather simple mathematical models that do not require precise knowledge of mast ofthe molecular details. These molecular details are instead chunked int system-level parameters, The models include only isformation on whether X ast ‘ates or iibits Yan at what activity threshold la these models, loge input fanetions can be used wv gain a back-of the-envelope sketch of the behaviour of diverse circuit, aided by the graphic fulton of piccesese- exponential dynamics that cross activity thresholds. Simplifice models ave particulayly useful for schexsatically deducing dynamical behavior and is qualitative dependence on biochemical parameters. This analysis can readily be experimentally tested, by using con trolled experiments that keep inany parameters relatively constant, aad dus approach tne \Weatize situations described bythe models Further re, de same mathematical wodels often apply to different networks, For example, paxluction-degradation equations describe gene expression dynaunics in tra scription networks (Chapters 3 195), kinase activites in signal transduction networks (Chapter 6), and exca sinaple mado of neutoos (Chapter 6) thus, simpli! models cus conn motifs that workin lfrent networks on dillereat timescales, ‘thie fevel of simplicity is the conceptual shmilaty of seeming a similarity express in terms a unifying es rebusties to component factustions:& biological system cust wir wader al pssie vt itorfrences that cone with the inherent properties ofthe ewmpanests al the environment, thus, #, coi needs ty be eobust with respect tu temperature changes ees and cick i the cll shuld depen! oa having prcesehy snd not 108. "The fet that a gene cieuit rus be rabust to sch pertarbatins imposes severe constraints on its Lesign: only a small percentage of the pose nce mvs poss sbustess ean help theoests to recogoize the correct mode ‘We examined specific examples of robustness it bacterial cheinotaais and in embry onic patieing in Chapters 7 and & There ate several ways wv achieve robustness For example integral feedback cas pro prinples One such desig pei cover a fee tens a 100 copes of protein X sii cineuits that pxforn a giver Scion can performy it robustly ‘xochanisins ace ne robust, Vide bust adaptation It ca lead the outpat ofa system to & desired goa in the face of wk vara 7 Kntegral feeb eamploys a negative feeinack ral over the diffrence betwsen the ateal output and the goal. Ite in the environsient or the internal parameters of the system (Chapter sna poportional 0 the tine inte I eealback can be ‘shown in many ens t he a wnigue soktion to the problem af robus! adupiation in the conte of engineering conte theory. Biologieal systeris ean seadily implement inteyeal(eedback because integration over time ig inherenfaruee of the producti + degradation equations mentioned above, ‘which describe may ofthe biochemical interactions in the cell. We saw how integral feed is implemented in the chessotaxis system by means of accumulating methyl tion that regulates the activity of the chemotaxis receptors. In ath systems, acc ing protein levels o protein modifications can play the role of the integrator to Fntgral feedback and robust adaptation. ‘An additonal principle of robustness in spatial patterning systems is selfenhanced egradation ofthe morphgen, as we saw in the case of fut fly development (Chapter 8) Self-enbanced degradation allows long-range pattern fo ependent fof the production rate ofthe ssorphogen. This principle appears tw be use agin and ai in iflerent yltersing systems with ferent morphogens. ‘A thind general principle tht confer robustness is kinetic proofteading, a mechanism that allows molecae detection of «specie molec rapt the kro sige of hemcaly similar molecules in the cll (Chapter 9). Kinetic proofreading relies on time delays that eas be implemented by diverse kinds of nonequilibium seactoas, The sante principe seems toappeae in systems ranging from translation in the ribosome to antigen {detection i the in mune system, Al! ofthese design principles ite biochemical deals that right otherwise appear as wastefil side reactions. ‘The few ways to achieve robustness help to Timit the numer of possible crv in biology, and to give the clscits that do appene a defined sil, His kel achieving robust designs twill be discovered that cat site our undersaning of diverse systems (Kita, 20045 ‘wagner, 20086). ion thts early lows to waders 1 aditional general ways‘Uroughout this hou, we have used engineering rictaphors andl gained inspiration Sor» engineering principles, One example is the consideration of response time and ta Vility in autoregilatory Feedback loops (Chapter 3) and ather network sot. Stability ane! response tine tade-ols are ofthe essence in electeunic al miechanical engineering, slesgn. Aawtbr point ofsilaity is the principe of robustness to couyonen! lel tins. Robustness is a guiding prineple in engiveerings far example, electronic ‘work despite variations in te resistance af each resist Lrigineering textbooks are Site! with robust designs, and many alternative nontobst designs for the same circuits se avoided Good engineetin wes modularity ad securting circuit elements (network motifs) 10 build yeliuble al setae devices from simple subsystems. Anadditonal ste ‘larly is optinal desig, with cost and benefit trade-off, which we saw guides evolution any selevton in simple system (Chapter 10} Such cost-benefit wakes ace bigs Sn enginsering. I addition to the simiacities with engineering, biological circuitry also has fund rental conceptual dillerences from engineered devices, One important ference i the stochastic nature of biological function. Hese, can that genetically identical cells ia ‘the same enviconment respond in probabilistic way toa given stimulus (ee Appendix 1}. Often, the response of each cell is not predictable, but the proportion af cells with 8 given response fs predetermined ane! i sulted! according, to envito ental signal For example, we saw that swimming bacteria have individul chatacters and perform chemotaxis with diffsvent bling rates and adaptation times. More dramatic examples Include cellar decisions such as differentiation or cell death sn which a faction of the «xls assunic ome fate and other cells. a comely ferent fate: Evalution appears te select fara probabilistic outcarne, An clement of randomness in behavior is one of the most famiiar features of tving ‘anisms In contras, most engineered designe re mace o try and voit stochastic ex ‘comes. Engineered devices such as rao ae designed to Faction with 100% probability 'G say, the ON bution is pressed, We would say tha probabilistic radio isa malfunction: ing radio, Stochastic designs ate not normally found in many areas of engiacering, but they are common in the fils of game theory and econantes game thr. iam sometimes be shossn that proluabilistc strategy is aptinal sen competing wit other organisa A sleterministic repose wuld be easly exploited by canipeitors, The sochastie nate Df cellular responses bnoudens the range of possible responses in an unpredictable Tatu, ‘nereasng the probabitity that at eas feaction of te cells wll beable to cope with sud «lon uoforesten changes in circumstances. Sly ofthe poe of probabilistic Resign in bio: ‘ogy is only at its beginnings! ‘An additional ineigaing set of questions concerns the interplay between the ecolngy and the biological design ofthe oxpanisi, We currently have more information the etailed structure of biological cies thas on the enviranmneat in which they evolved We know litle about the constraints an! functional goals uf ells within vampex org sss andare ony beiningteunkrstand he opts rie mei thi design isan nesting question wether wuld be palo frm they of Blogia ei a con elp ify spt of ely eatin, lel Seg. "We are sont the end ths bk, wh ri fo pee a tradi 1035 tems ly tits im aly wre inten, sine eee a Te ‘poning athe venture fo Gnd the design pence of Bos systems. a this Don haere to enhaste simply mB, tn sid comps, encourage the apts point af we ta gene prncpescan be covered. Who suc prinepes tif imaginhow we can mak sans of gy om thee of mente a ate, or ogi Wills complete dsp ofthe igi networks ofa ete cll or ergonsm ce a he ofan hon eo nn neering, Mich of ngneringis actly ere cing, ens rteyper oto tot wean ned web understood crt hd a py isreverscenponringo a rt cle, There ean dies to heaven thi projet Rovere engnesing os nomodeh highly wre taro ee hoe Sand components and txt notes intention evita impose However, the Simpityng ature tht wha dics give hope tha aga eto ore tues ht man beings ean understand Madly, fr sample tthe oot oF Diy te separate he pole inte smal is that an be aed pent pend Tosi facts to Bee rotsin, al pathway and wo on The princi or nes isthe ange of posible crits tht Tcton on paper to ony 3 Tew dss thc wo inte ell Tn coher to hme the cnet dein wth Foe at Nerwore moti fine the fw Dose pater ha ae up aatork and the ictamary oeemennry dynam anctineta he nt can perro, These concep ogeter with the current ecole reveksin in ky, May evenly Slow haat and undctaing of we network, wth ret ene tren. The sili been the cents evelaton a nose ah ass a fanamenal semi calnge' understanding the wf nator that wie ved and esi stomThe Input Function of a Gene: Michaelis-Menten and Hill Equations re Ad BINDING OF A REPRESSOR TOA PROMOTR “This appendix poviesa simplified mtcoduction to basic models in biochemistry, We will begin vith undeestardng the iteration of a repressor protein with DNA and with is inlucee! We will then tur. to activator proteiss, The eepressor X recgaizes and bil 10 specific DNA site, D, ina promoter: X and D bil t0 tion ofthe gene ueews only when the repressor is no! bound, that is when Dis fre. the DNA site ca thus beeither fre, arbour, [DX, eeslting in conservation equation: sa complex, [XD}. Transp: Deixb]=p; wu where Dy isthe total bacteria cell moans th concentration of the ste. For example, a single DNA binding site per Dy = cell volume ~ tun? ~ 1 nM. in eukaryote eels the vol ume ofthe nucleus i the ner of 10-100 The epressoe X and its tanget D diffe in the cell and occasionally cole to form w comsplen IXD. This process can be deseribed by massaction knclies: X and 1D cote “Ind ind each other a: «rate Ky» The rate of complex formation is tas proportiona so {he cllisin sate, given by the product of the concentrations of X and D: rate of complex formation =, XD 2aBaz & APENDIA A {Use samples [XD alls apart feissocistes) a Ye by, Ihe rate of change of [XD] based ‘om these caision aon dsvtaion procenes is described hy SIX YIM XD = ke LD) (aay the rate parameter fr the collisions, Ky deseribes how many collision events ct por scsi por protein aa given concenteation of, an! thus as units of Himelconcentrs tion. Is useful to cemsember that k,n biaelimical reactions is often lite by the sf gollisios os difasing molec hitting proein-size target, ad has diffi ited valve of about h, = 10° 10" sec andependent of the details ofthe reaetion the case of a transcription factor aod DNA, the dis lint is usually ger because of ‘oe atnenstoil cus elects due stg Ue auction fate shang the DNA, y= 10 1H sec Berg etal 1981}, the orate kyo the eer and has wits of Une and can vary over many oners of magnitude for diferent reactions, because hy is slxermined by the strength ofthe earaieai bonus that bind X and D, the kinetics of uation A.12 approach a stendy-statein whieh concentrations dk not change will tine lXDVAt~ 0. Solving Equation A22 at steady-state, ne find tha the blaice betwen the collision of Xan D an the dissociation of [XD] leads to the eh «al equilibrium equation: K, [xb] = xD. wus ‘where Ky sth dissociation constan Ky hella te dissociation constant Ky bs anlls of concentration. The larger the discon conta, he higher tbe rate of dissociation of the comple, thats, the wesker te binuling ofXand D, ‘Slving forthe concenteation of Fiee DNA wes, D, uss Find Ky (Dy - D) = XD. which yetds Equations, AD and ALS. we blo b, 1exA waa For many repressors, [XD] complexes dissociate within less than 1 yee tat is > 7 sect Therefre, we can average over times much longer than 1 sce and consider Di the probability that steD i re, averaged over many binding and unbinding events. Ihe probability thatthe ste s Fee, D/D,, is decreasing funetion of whe concentration ‘of repressor X, Whi ther eo repressor, X = 0, the site is always fee, FVD hhasa 80% chance of being fre, DAD, = 17, wien X= Ky When site 1) is eee, NA polyonerae can bind the promoter und tratseibe the gene the fale of transription (owmber of MRNAS per second) from a fre site is given by the miasimal transcription rate (Note that sn the mai teat we used fs to denote the rate of protein prolaction, Ts rate 8 proportions tothe trassceiption rate times dhe The site ax GUE A Serna promoter atriy opetar cncetaton nai ofits Ry Ha main snoceuce ea Kc the ine, Hee Xcess ote conenraton a Fees ste Foon number of proteins translated per mRNA provided that thers isa constant mRNA Bile- tion rate depends on the DNA sequence ‘and postion ofthe RNA polymerase binding site inthe promoter and other factors, ttean he tuna y evolutionary selection, for example, by means of mutations that ehange the DNA sequence of the RNAp binding ste. Tn different yones, [ranges over several orders fof magnitude, B= 10*= 1 mRNAs, The rate of MRNA production, called the promoter activity, times the probability that ste D is free: ‘meand translation rate) The maximal trans promoter activity (ars) Figure A shows the promoter activity as a fupetion of X. When X is equal to Ky transcription i reduce! by 509% fom its maximal vale. The value of X needed for 30% ‘maximal represion i called the repression coefficient Fur effcigot repression, enough repressor ie needed 30 that site D is almost always ‘occupied with pressor. From Equation A.L, thie occurs when repressor concentration ‘really exceeds the dissociation constant, suel that X/KK,>> 1. This i the ase fer many epresors inching the lac eapressor Lact So far we discussed how the repressor binds the promoter and inhibits teanseription “To cura this gene system ON, a signal rast cause X to unbind from the ONA. We will cca the simplest case in which a small molecule (an inducer) i the signal. The inducer diveily binds to protein X and causes I t9 assume a molecular eanformation where it ‘docs not bind D with high anit, Eypcally, the affinity of X to ks DNA sites is eeduced bya factor of 10 to 100, Ths, the inducer Fres the promoter and allows transcription of the gene. We now consider the binding of inducer to.HURT At Hoatom aX tourna fins 5, conan, Half 0X ish whe, Ky AZ BINDING OF A RTPRESSOR PROTEIN TO AN INDUCER: MICHATHIS MENIEN FQUATION i ‘The Fepzessor protein X is designed to bind a small molecule inducer 5, which ean be conskdered as its input signal. The two ean collide to form a bound complex, (XS,] The epressor is therefore found in ether fice form, X, or bound form, [XS,], and conserva ‘ion law states that total concentration of repressor protein is Xe x, xl d 20 X and 8, collide to form the complex [XS] ata ate ky id the compl part (dissociates) ata rate Thus, the mass-action kinetic equation is xs, falls PKS, = By XS, ~ hg (XS,) (ara) At steady state, €1X5, 4 = 0, and we find the chemical equilibria eelation K, 0s, (423) where K, isthe dissacation constant forthe lac repressor, K, = 1M ~ 1000 indacer (TG) moleculestel) Using the conservation of total repressor X (Equation A.21), we ‘rrive ata useful equation that recurs theoughost bislogy (this equation is Known as the ‘Michaelis-Menten equation in the context of cieyine inti; We use the same name 1 the present context of indvcer binding} XS Sak, ‘esi X pcos htt bred Boot "say ote on ch gt nt mes eae 9 wee ry stoconerten st y= 8 er otamge nla optn teae Ta sane ide evar ft fens, “Houma kin smash ei Sits dew “intent tea pw Ky = igh in Meine etc Mog an xs, ‘Michaelis-Menten equation (4.2) le ty = HMMS i the THE INPUT FUNCTION OF A GENE @ 245 The Michaelis-Menten function (Figure A.2) has three notable fates 1. tereaches station at high S, 2 thas a rogine where (XS, increases lineusy with S,-when §,> K,} is known a5zer0-orderbecavse [KS] ~ 5, and the linear regime (5, c K,) is known a5 first-order since [XS] ~ 5, Recall that in cases lke Lc, only X unbound S,, is activetn the sense that itcaa bind ‘the promoter D te black transcription. Hecause free X is active, we denate it by X* Active repressor, X= X,~ [X8,, decreases with inctessing inducer levels x, Tas my MeN of X ot bwnd ws, (028) AQ COOPERATIVITY OF INDUCER BIND- ING AND THE HILL EQUATION Before returning w the input function, we comment on a mre realistic description of {inducer binding. Most transcription factors are composed of several repeated protein sub "nits for example, dimers or tetramess, Fach of the protein subunits can bind inlacer ‘wolecules. Often, fll activity is only eached when multiple subunits bind the inducer ‘useful phenomenological equation for this process can be derived by assuming that n molecules of, ear bind X, To deseribe the binding process, we need co describe the binding ofn molecules af, t0 X. the protein (protin mukimer) X ean ether be bound to m molecules of, described by the complex [s, XJ, o¢ unbound, denoted X, (in this simple treatment, termediate states where fewer that n molecules are hound are neglected). "he total concentatian of bound and unbourd X is Xpand the conservation la i (08, X] =X, (aan ‘The complex [a X] i foxne by collisions of X with n molecules ofS, Thus the rte of the molecular callisions needed to form the complex is given by the product of the concentration of fee X, X,and the concentration ofS to the power nthe probability of Finding copies ofS, a the same place atthe same collision rte = hy, X 5," (asa) here the parame, describes the on-at of complex formation, The complex [aX] fissoriates with rate ky dissociation rate = hy fa, X] (3a)246 @ APPENDIX A Ahhh he Hil ecient amauta indo ocure 8, - Ky The parameter ky corresponds to the strength ofthe chemical bonds between 8, and ts binding s tes on X. The total rate change ofthe concentration ofthe complex ¢ thie the dilference between the rate of collisions and dissociations: lls, KIA = eX Sy ho iS) ase iis equation reaches equilibciunt within milisceunds for typical inducers, Hence, we «can make steady-state approximation, in which dln, X= 0, to find that dissociations balance collisions kuin a3) ‘ie can now use the conscrvation equation Gquation 3.1) 1 replace X, ith Xy - Ins, xh to find plece X, with Xp (aol In8, X] - OX; ~ [a8 XI) 8," (AS6) Finally we an sole er th ation of und X, find nding estion known as the Hill equation: ee [nS,X)_ St Xe RS siete neve defined thecontant sich that Hillequation — (A.37) (43s) tow 4.37 can be considered the many binding and wnbinding events ofS, bability thatthe sie fs boul, averaged over TE INPUT FUNCTION OF A GENE #247 “The pucameter 1 is know a8 the Hill coeffckent, When 1 = 2, obtain the Michse lis-Menten equation (Equation A.2.4) As showen in Figure A.3, both the Michaelis-Men- tem anil il equations reach half-maximal binding when 8, he steepness of he Hill ores greater the larger te Hil the lac systems m= 2 with the inducer IPTC (Yagi anal Ya boy Hill. coolfcentsn > 1 are often termed coopera “The concentration of wabound repressor X is givea by cient (Figure A.) Reactions described 1 GT or AA WILMONOD, CHANGEUX, AND WYMANN MODEL _ legant analysis of cooperative binding based on syn ‘We note that @ more rigorous 2 metry principles is due Monod, Changeus, and Wyman, ina paper well worth rea fing (Monod et a, 1965), usually also described in biochemistry textbooks. In this model X switches oan active state X" and back. The sigaal 8, binds X with dissociation constant XK, and binds X* with a lower dissociation constant Ky". Up ton molecules of , ean bind 0X. The two states, X and X* spontancously site sue that i the absence of Ss Sound at polity Target by B-han The resus: Kasi XL TOS KY HOS, 7K Antesesting extensions to this model make analogies to Ising moxils an physi (Duke tal, 2001), One difference between the rigorous models and the Hill eurve i that bine: Ing at low concentrations ofS, sHneaeinS, rather thona power law with coefficient 1 in Equation A.37. is linearity is dc tothe hinding ofa single ste on X,zather than all sltes at once, JAS TE INPUT FUNCTION OF A GANE REGULATED BY A REPRESSOR ‘We can now combine the binding of inducer to she repressor (Hquation 4.2.5) and the binding ofthe repressor ta the DNA (Equation A.1.4} 0 obtain the input function ofthe ‘gene. The input function in this case deseribes the rete of transcription as a function of the inp inducer concentration casa) Figure A.4 shows how the transcription rate of a gene repressed by X increases with increasing inducer concentration S,, Note when no Inducer is preseat, there i Keakage transcription rat, 8, = 0) = lt + X;fK,)y also called the basal promote Teakage is smaller The stronger X binds its DNA site. ‘he inp fanetion reaches half-maximal value a inducer concentration S, alfa induction point is approximately (hen X22 Ky)sh HOT fat fone eta transect na fctnn fier fr ayo pate by eres Shows ae RyK,= I per and XK (tony are, eth win = Tere i lehage ‘coservonat = and harman tection reaches, 3 Ke and S07 Re Sia OG IKK, (asa) the halfway inducer cancentration Sy, can be significantly lagee than K (Figure A). For Lach, for example, XyfKy~ 100 and n= 2,50 that §, =10 Ky We now turn to deseribetransception activators, Ab WINDING OF AN ACTIVATOR TO ITS DNA SITE Tn the decade flowing the discovery a the lac repressor, other gone systems were found to have repressors with a similar principe of action, I is interesting that I 10k several years forthe scientific community to cept evidence that there aso existed transctip- onal activators, Am activator protein increases the rate uf transcription whem i binds to its DNA site inthe promoter. The rate of transeviption is thus proportional to the probability thatthe octivator X is bound fo D. Using the sane reasoning as above, the biting ot Xo D is Aeseribed by a Michaelis-Menten function: promoter activity aon XK, “Manny activators havea specific inducer Sy such tht Xs Functional (in she sense that it cam bind DNA to activate transcription) only wien i bits S,° Thus, we obtain "Insecta act inactin eatin San inv whe shown sniniar of Sovbp sone roreans vt be ee ‘ingthe vega th app tc ene ing Tee cnc be eal esc THE INPUT TUNCTION OF A GENE 249 [HCO A put finction ora gene tga yan sctvtr ata function ofthc Ir Shown sete carves ar NR» 10 (butt curve) 29d XK, 3p cure) beth whe «2 The no beat Transcription ar 5, and amano action each = 19 Kean > WP XS HBS) EES (062) “The genes input function i AS) = BXHK, +X) 63) “this function, shown in Figure A.S, is an increasing funetion "The basal transceip- tion level s zero inthis regulation function, fs lower leakage than repressors. needed, hovsever, a nonzero basal level can be really achieved by allowing RNAp to bind and activate the promoter to a cet the absence of activator ) =O. Simple activators thus cam have nextent even in ‘The inducer level needed for hall-aximal induction of an activator can be much sole than Ky Sur KIX DK, (Aa) in contrast to the repressor case (Equation A.52), Overall, however, similar input func tion shapes asa furcton of inducer S, can be obtained with ether activator or repressor proteins. Rules that seem to govern the choice of activator oF repressor fora given gene are discussed in Chapter this appendix we described a simplified model that captures the essential behavior of 4 simple gene regulation system, in which proteins ae transcribed a a ate that increases With the amount of inducer S, Many real systems have additional important details that230 4 ADPENDIX A ay ax 06 medic we hi et oon, nahin ete oe sake then jghter and sharper switches? The present description Issificient, however, to ‘understand basi cre elements in transcription networks. Ate Chonyparions of Dynansies with hagie ani pat anctins Hove good i the approximation of using logic input functions (ce Section 2.3.4) insted ‘of graded functions ike Hil fonetions? In Figure AS, the dynamics of accursulation of a ple cme-step ranseription cascade at shots, using theee different forms of the impor function fX). the inpot finetions sre Hall fanetions with n = 1 aed ry me input function. At tvs t 0, X* slats to be produced, and ils concentestion inezeases igradoally ith time, The graded input factions show expression as soon a8 X° appears, ‘wheres the logic input function shows expression only when X* crosses the threshold K ‘Overall, ne dynamics inthis eascade are quite similar forall three input Fanctions and a logic AZ MICLIAELIS- MENTEN ENZYME KINETICS, ‘We now briefly describe a useful model of the action of an enzyme X ots substrate S, to catalyze forovation of product P. Enzyme X and substrate § bind with rate Ky t0 form 4 complex [XS], which dissociates with rate . product P.so that, ne complex has 9 smell rate to fort (any the ale on fr hig in coun he scan of Sino X ‘well as into X + P, is. * Stine XS > Kyo BEO> at which the production rates half maximal. When substeate is sate ‘Toction Isat ts maxiina rate, equal tv X, Thos, the prodaction rate doesnot depend on ' (that it depends om Sto the power zeo) and is known a5 zero-order Knees: production rate = Xy -sero-oner kinetics (ary {hn the main text we will sometimes make approximations to this function, im which the substrate $ is found in low concentrations, $ KKB, U1 90> Ky] +B, 00¥> KD) O16) where we remember that @ isthe step function, equal to ort These results ave so ratio of polynomials oft generality. The input fonctions an ofien be described by the active concentrations ofthe input transcription fctorsX 4 JTIADIMENSIONAL INUUT PINCTIONS #255 Sper iSoumi ‘The parameter K, isthe activation or repression coefficients Fort Bis ts maximal contribution to expression, and the Hil coeliiens ar tars and n = 0, m > Ofor repressors. These types of fonctions have been found to describe txperinentlly determined input functions (ety et, 2003). More complicated expres: Sions ae possible if the different transcription Factors interact with each other on the pro tein evel (Bucher et al, 2003). Mp Xe) = wean cripion fxtor X n= for activa LXIKLISs Th The prone ig enugh eto Xan ees Yi promote the Teper anl eat tes oeps tht X and ¥ sana bah BME at he sae tie Whara the suing inp fet? How dos die on the np fone Nom obtained rm indepen Bing?svonsoxe C Graph Properties of Transcription Networks CdL__TRANSCHIPTION NETWORKS ARE SPARS What is the max xT number uf edges ina network with N nodes? Fach nude ean have an ‘outgoing edye to each ofthe N ~ 1 ather nodes, for a total oF Fay = NCW ~ 1) eles. I we also allow self es, there ace an additional N possible edges, ira total of f= N¥, Note that @ maximally connected network has pair of edges both directions (mutual edges) between every two nodes. Ue number af elges actually found in ivanscription netéoeks, Fs mach smaier than the maximum posuble nuenber of edges, The networks are spars, in the sense tha E <1 Typically, less than 0.1% of possbleeages are fon in the network. Uranseriplion networks ate the product of evolutionary selection. Its portant to rote tha ie very cay to lose an edge in the network: » single tation in the binding site of Xin the promoter of ¥ can cause the loss ofthe interaction, ‘Therefore, every edge in the network is under evolutionary selection ‘The sparse nature ofthe network reflects the fact that only very few and specific interactions, with useful function, ae selected and appear in the network, C2. TRANSCEIPHON NETWORKS HAVE LONG-TAILLD QUIPUL DEGREE SEQUENCES AND COMPACT INPCIT DEGRET SEQUINCES ‘We saw that nodes inthe transcription network correspond {o genes, Incoming edges regulate the gene, The wumber of edges that point into a node i called the nodk’sin-degeee, the out-degree is the number of eds pointing out of node, corresponding to the numberof genes regu lated by the tanseription factor protein tha is encoded bythe gene (or operon) that cor responds othe note. toa node in the network correspond to transcription factors th 2the mican number of edges per node called the mean connectivity ofthe network. is = HIN Typically A ise the order of 21010 edgen/nad, 1 al noes have si se dees? Transcription networks almost alaays have nodes that shew sac h hgher vat degrees dn the average node, Transcription networks often sve many transriplion factors thal rege fer genes, ever nodes that regulate tens of ste and coven Fower that reglste hundreds of gens, Ihe ater ue called global regula. tors and usually respon o key environmental signal to conte large ersembles of genes (examples of global regulators bm bacteria include which responds to glucose star vation, andl Rpos, which responds to general stzeses), Thus, the out degree distribution Inu a Fog cal and can be roughly described as 8 power lw a lest vera certain range (Garahasl and Oliva, 2004). Thot is the sumer af nodes with our degree ks roughly PQ) ~ les with y ~ 1 to 2. Note thatthe ont degeee distebution i only approximately povser lw for example, its bounded by the numberof eres The longtaed distribution is sometimes called “sealefse” house there are sets of late genes uf many diferent sizes with no typic scale, Nodes with many more co nections than the average ate called hubs. Hubs are Found in many types of natural and cenincered sys Ih vs, The question of thie origin in biological networks isan interesting, otrast fo the long til ofthe outaegree dstributinn, the in-dege distribution is concentrated around its average vale! (Ihietty et al, 1998; Celina eta 2002; Shen Ore etal, 2002). The in-aegrees range between zero ada few tines the mean eonneetiv ity A. Thor is ile chance of finding a onde related by 10 a¢ 100 tines more inputs tg the average node. In other wands, the in-deyrce distribution does wot have @ fang tail, and instead resembles compact distributions such as the Poisson dlseibution, whase stanidand deviation is about the same ashe mean. ‘he compact distribution of in-dogrees may correspond i part to a physical itation, In simple organisms, promoters are shoe. The ragon near the RNAp binding site that par tiepates in regulation ison the onder ofa ew hundred Base-pairs(DNA leter). Tere is spc inthe promotor regis to accommodate mare than af bing Stes for transcrip ton factors feah on the onde of MI base pits. fn sone complex organisms, eransexip tion fetors can alot gone «ven if bral Far aay on the DNA, throwh PSA-Laoping Sony saad ginal atm hn a se ut Hy hae ern inns ype of wetout pne cho for gain pret a Ate oe Inte mal ele eer tae uw ete aed a ant wth high ite many canactr, Is acer poate ets whe fee sical ceed am nen hg wd sini wih pct toe inl egret on anc mee He nt fe oa om tert ti Wee tg gil es Ihnen ta hat nb pay wih FPretlusi dogpetar aye spe sly ee bac or et appt hy nko aad eg 3 ‘nine ei reshch he sol he le eat bas ey pron tc of oo by Be GRAPH PROPIRUIS OF TRANSCRIPTION NETWORKS 259 fnteractions and other effects. Such action aa distance can inerease the numiber of mpd igher onanism alten display larger in-eyrees tay he comples computations needed during developsient transcription factors toa given ge microorg ps, aecomnedan 63. CLUSTERING COFPICIENSS OF TRANSCRIPTION NEEWORKS [An additional statistical peaperty of graphs is the clostering coeficient, which corre: spor to whether the neighbors of «given noe are connected to each othe. Het ws con Siler the network as nondirected that is, diseegard the direction of the eyes. A node tik neighbors can be apart of at ost k(k-~ 1/2 thangs, one foreach possible pat of neighboring nas, the elustering couicient Cis the average mumiver of iangls that a node participates in. ivi by this maximal number. Vranscription networks have aver Age clustering coeicients larger than those of randomized networks ‘As descebea in Chapters 4 through 6, network motifs in seusory transcription nc works generally ince one main type of tiangl, the feed-forward loop. The njoe Contribation tothe clstering ollcien! of transcription networks therfore stems from feed-forward loops This pater appears to be scecte due to is functions, suchas filter- sng and response aceleration ‘he clustering coeticient can also be measored as 2 function of the number of neigh bors that each node fas, resulting i a clustering sequence C{K), Often, C(R) ~ UR over a certuin range, so that the more nxighbors a node has, the lower its clustering coetiient (GBarabasi an Ole, 2000, In transcription networks, this tendency appears to vorre sporal to the way tht feedforward loops connect to each other. The chief aetangement of feed-forwant loops in sensory tomscripton networks i the multi-outpat FE, discussed Sn Chapter 5. ln the mult-outpat F1, node X regulates (aid is thus neighbor of), and both X and ¥ regulate & output nodes. These ousput nodes are typically not neighbors Thus, node X has k + ¥ neighbors (Y and the k output nodes, with only k commestons between these neighbors (the conections of ¥ to the output), esulting ina cistering coolficent C~ Ky Wk, Generally it appears that global statistical properties of biological networks such as degree sequences and custering sequences are the result of seleetion working on the ‘etal eiruit patterns in each individual system, Dillerent netwotks have ilferent selec tion constraints, which must be understood in arder to understand their graph properties C4 QUANTITATIVE MEASURE OF NETWORK MODULARITY [Network modularity i the degree Io which it can he separated into nearly independent sub-networks. A quantitative measure of modularity was developed by Newinan and Gie van (Newnan 2004; Newinan and Girvan, 2004. Briefly, the Newman and Girvan algo rithm finds the division of the noes into modules that maximizes a measure Q. his rncese eefined by the fraction ofthe edges in the network that connect between nodes e quantity i a network with the same ina module minus the expected vale of the sin assignment of nodes into asadles but random connections betwen the nodes«ay there K is the nuniber of modules, fis the number of edges in the network. fis the number of ees betwen eles in modules, and dis the sum ofthe degrees of the nodes in module s.'Ihe eationae for this modularity measure i as fellows (Guimera and Ama 1.2008 gl partion of network ota moles mst empese any ule ed rn ny cs fw pabl nen: edges lore yo ins ‘the number of between-module edges {or equivalently maximize the : ber of within ‘ove edges), the optimal partition consists ofa single module and no between module exlges. Fquatinn C1 addresses this ditfiulty by inuposing Q «dom ito modules or ial nodes ae in the sante module if modes are placed a ran This measure can be further sefined by normalizing it with respect to randomired net works. The normalized measure Qy is (Kashtan aid Alon, 2005) Q=,-9, Qn,“ Qya) 2) where Qu he Qvaie af the ewok, Qu he serge Qvale of randamivd ret and the mail posible Qvale of eter with th same degree teqoece 5 thee network The vale Qu Quan MQ. can Be clued bye Gent aoithns (athtan ad Aon, 2008) they tsnve of maiy mets out the ets of network te ae sci Bisogcalnetwork sw ig moda acordingo this menor Teo son vt cer hrs th mn sae network of the nematode Caenorhabditis elegans has Q,, = 0.54, and a human signal trans- duction network has Q., = 0.58. SarsbesQue0Seanteh teal H Cell—Cell Variability in Gene Expression —_— ‘the concentration ofa prtein X én popalation of genet call to cell due to stochastic processes (eviewed in MeAdams and Arkin, 199% Kacrn 1, 2008), The concentration of a given protein often has a coeicent of variation (stan {ard deviation divided by the mean) in the range CV = O41 fo 1 (Elowite etal, 2002: Ik etal, 2002; Hlake etal, 2003; Raver and O'Shea, 2004), "That i, the cell-cell nas aren the pier of tens of percents ofthe mean. The dynamics of protein eves aly identical calls varies from thus havea stchbstc eumponent (Figure DD. ‘Gue impoctant source of noise is extrinsic noise, in which the cellule capacity 19 pen luce poets, and the regulatory systems that regulate a gene, Suctuate over He. For tesarpl, fluctuatiens in & transcription factor concentration can affect the expression tate of is targets. The correlation tine of these variations in product the seale ofa cll generation: that i cll with high production levels often tends to stay high fea cel eycle or moe (Rosentelt etx, 2005) in addition to extrinsle mbise, there isa Fteinsi noise, which is due to stochastic variations inthe tenscripton and translation evens ofthe yene. An clegant experiment by Michael Elowitz and colleagues (Howitt al, 2002) measuted the euatve level of iieinsic and extzivsie noise, by measuring the levels of two Buorescent pootins expressed by identical prometers (Figure 1.2). tntrinst aoe appears to Muctuste om a timescale of snintes in bactera (Rosenfeld eta, 2005). “The cell-cell dribation of protein numbers is often similar to lognormal (a Gaussian disteiution in the variable log(X)}, Whereas Gaussian distributions deseribe processes that arc a sum of random variables with finite mean and variance, lognormal distribu tions characterize processes with several multiplicative stochastic steps (because log) is then a sum of rantom variables). Examples of multiplicative steps in the production of a protein are transeription and translation,262 APPENDIX O Protein Carcerraen | Cea canaaten CURE Sheed a tn anti A ale pt {oll ninting poston ste The cing damier show Hatton eth sb and st se ake" jie eoning othe dtm me (etn 2433s sown ight CO Nomateeamein CFP any LHCLURE 1.2 A enone aeromedical one tao prin wit i cat Avorescent cobirs, yellow and cyan Auneescent prot mit and CHP) The smut noe el ma one amen oe SS us a ce Sha he ep niin an eee ose prc veon sll recap oem Hew ey 2002) Regulatory circots can affect the variability. For example, protein Jevels can be made to flctuate less by means of negative feedback Toaps (ae Chapter 3). Con sey. psi aucun cm itr lvl, Song pie fd ack can even lead to bistability (Figure D3). Bistabilty often leads «a bk-modsl CELL-CELL VARUAMIILY OS Gem Ear R tae = calls sve suorsgution ca goealy decease CURE bs Disdaton of pesein munbers pe cal Ne ation ac es oa Ba Mae ee ue date Poss autoneolaan wie the st Mra cals Tos ipatens par cal) ACUI Sehematc darts of protein conection in argu cascse, Vari tends to snecase wath he stp in he case, “istrbution, with two cll populations, ith high and low expression (Nuwvick and Weiner, {9st siegee and Hu, 199%; Ferell and Machleder, 1998; sans el ah, 20075 Orbudsk ct 3h 20n2} Nowe ean also be ampli by regulatory easeades cach step inthe cascade atv variability From ts upstresun regulator (Figure D4) (Blake et a 2003; Tooshang rey e005, and Pedraza et al, 2008). Rapidly degraded proteins can have narrowertistibutions thas stable yroteins, because stable protsinwinteprate the noise ia produc: tion rates over longer times. Asa role of thumb, the fster the respnse tne ofa system, ‘he smaller the Muctuaians sn the syste ‘One interesting observation is tbat tbe postion af the nuisiest step ina pathway ean vence the overall noise (McAdams aod Arkin, 1999; Orbadak et al, 2002) This is Fheatse each step in the pachwuy usually amplifies noise in the previous steps, Foe exau pl, consider two mechanisms that prodiace 100 proteins per hove In mechanism A, one MRNA molecule is made on average per hour and is translated to 100 proteins on aver. gel mechanisen 100 miRNAs are nde pe hour and are vach eansbated to one pro tein on average ‘the Muctazons i proicin production are much lager in mechanath Ay because am average of one mRNA norrallymicans that in somie cells either O or 2 mRNAs wal be made in a given hour, resting in © oF ~200 proteins. tn mechanism B, there is wo make reco mRNAs during a hous, and factuations are smaller “the chromosomal position of «gene ean alo affect aise, de to local differences in cliromatin reglation (Blake eta, 2003; Beesk' ets, 2005), Generally mise level can be ‘uned over evolutionary timescales by changing the parameters of the noisy steps in the ceapression ofeach gene (Praser etal, 2004) I appease that essential proteins and com: lex Sorming peoteas ae less noisy than other prteins ite chase ‘Noise in biological systems can be modeled wsing stochastic mathematical equations Such theoretical treatment of stochastic effects #8 beyond the present scape. Excellent lextson stochastic processes such as those by Gardiner and Van Kampen, can give access to the highly develope field of stochastic theory in physics, chemistry snd engineering ‘howry an biological noise has bees reviewed (Paulsson, 2004; Kaern et ab, 2005). Other theoretical studies ae cite in the bibliography. FURTHER READING. Acar, M. Bese, A. ail van Oudenaaedea, A” (005), Enhancement of elilar memary by edcing stochosteteansions Nature. 435228-232, Make, Wi}. Karn, Mb, Cantor, CR, ad Calin, J) (200). Noise in eukaryote gene expession Nate 422.63-697, loi, Mh Levine A, Sig. RD, and Sain, PP. (2002), Stochastic gone expression Sn a kal. Scene. 2971-186, ‘andi CW. 2064) Heth af hose Meth. Spring aecm M, Hon, EC, blake. WJ, and Calis |]-2095- tact in gene enpeesins fons thoore phenotype Natur Re. Gate AEA MeAdanns, HAL ani Atkin, (2997), Stochastie mechanisms i gene expression. Pros, Nh ‘Acid Set USA, BUA, Novick, A. and Weiner, M, (1957) aye induction as a ‘Acad Set USA 43 583-56, Ouludah, BM, hata, ML, Kure. L, Grosemnan, AD), and van Oudenasten A (2002, Regs Tatom of sen thecxpession of single gone Na, Genet 369-93. Roser. and O'Shea, EK. 2004), Control of sochasiciy in eukaryote yowe expression. So ce. 30481-1804, owerfel , Yung, [3 Mon, U, Swain, PS. ad Howie, ND (2005) Cine elation tthe Single-cell Sens 30762-1968 ‘r-none phenomenon. Pre, Nal Glossary Activator — A rnscriton stort nee the specific ae nthe ge’ promt Activation threshold ~ Concentstio uf actiatr i ie ative tat ect fr alana stir ‘hn ofa one ‘Adaptation — Desesing response aatinlus hat ap incl, ‘Adapuation tne Tine or output recover 9D of prestinl level lowing a step tinal Allele One oft of terative fern gone: Ina igo ganic sc tot ial ely cach yecls tall, one cco the (wo ar chrome, ‘Amino ackd — A deel ha cntns both ain geo NE) a9 earoxy group (COO. ‘Amino acre linked together y pepe ol seve athe nosis lyons, AND gate = Alpe fant ft inputs that coal itt iat ar al tonne Antsmotit— A tea that eur in nctwork Ka fen then expected ot dom, ‘Antibody — 8 prcn proce by acl af nnn system eso tesa poten presenti ‘ovinnng congas Antigen pact poten or ther elec hat scone by an anti, Arabinose—- sugar vive by Hcl as am energy apd carbon sores, ig the ane gene. these ‘ene nce metalic genes an AD, a the anspor ral sada P GE Arabi nt ppd ot he cel glove. a Better energy src, ps ACP (adenesne phosphite) — A mile tht the man eure in the ells enegy 6 ‘omy. Theoaverson of ATP ADP (adevonin phate) erates energy subi (ais subs) A acer canna foun th sl. rn Sle spore pn Binoovatdisteibulion — A stata estan th sce, fr example the probably ek heads ota teas of as hat hae probally to give sds and to ge lle Chetorceptor-—A recpoe that espn to the presence ns particule chen (Chemotanis— Meme up spatial yeionts of spec chaleur den gant spss chemicals epllers, Chromosome — i ston of DNA, with asec! proteins, Sou infra ‘Ciacadianshytbin A dlrs cyle cls atrty Generate by a buchen osc Ino ony cells in ania plat, ad ier The elton em he ‘entrained pete empesateaal it spats The sein rons aioe the saan of reining exter sully wih oid eset ier thm 249 aton “The cose esta ahh. ane speeds a ae a eet, A ‘Gavan These cae forthe 20 mi acd wih snl repented by mre th nevadon} thee af theron gpl tsa tpt fhe ten) overeat ed forward top = A enema In which ue et ah OX Ze ihe ssince thei the niet ath cn Xap 02 ‘Coherent patie A ptr with gos th ee ic, a ch paises he igh fal revedjthebtoeen Ihe the se of transcription ofa poe when binds nce caries puntie266 & GLOSSARY Cast-benait snaysie- & taory at seks de pteal design sah tha the dee ete the Sinamay synthe sacs ects Cytpta— hevcons sol bance has hd wi pro Degree sering rom pews — An ese of rated tw Vit hae the same She ane hee neni sng cp reach ah et) SStlcwal th left cape spa the some ety ef hah Succi tir samt chy ees ene ea the spr onlin aoe geting teeth tons ‘muy incitthenetwk rabies gi ee etd nathan fe se othe! ne nscale oe Desclopmentaleamergtin networks — Neral Wansrpion natin tha ie chang i cllpe porto snug tonto use the econ el fate cle {hit cnhep deca in rer Dewapmenl amp nets ek Te Shoes ofl gratin sl ten keds Thy sd coe! ‘ary tamcrinton cs at gee spent cont pal Diferentition-- The proces im which cB change ire tp fer. [DNA Goxyribonce ac) ~ Ang nec composd ft econ hel tran “Contin the echinacea the DNA ea Ee ya {ihe tw somber wharf pew ved © wi DA ade of cin of eps a everest ot ete Dorsal — Side of ar animal chaser to its hack, " " ‘Drona — Fry, ol ogra commonl us Gr gl rash age lnk ener woes anc. ie re ats bao heconpnents wale he en 8 el The ey bs keith by the woes Es naa tors have np rst, Mal es 6 edges tha Tink next both vei Se anerption network fora eae nocytons~ Upte a matralnoa eh nye A psn hat flats bck recon the ene ale the eset and cs nt ite cone prt ofthe and prod FR (Erde Regi evn networks ~ An emcembl fea networks with given mom of Boies, N and edges The dsr a placed antl betwen the nek. Is el ca Be ted fr camaro ore twos. A ove ingot anos sth dee ese ingraun atwon, Frrarlond =~ Te edo the cps bee dae ote ee aya, cli Ecerichi cll = A rod hp acter sel oa nthe taint Hiewidely std se organ Faukaryoi cll and organisns — Capi sade of cls wth a cles thal fos ie cep fr vires and bacteria (poate) Yea singel eakaroe organi beset alapttion (rece aplation)— A propery ofan aapting sensory sem in which the atedptae utp indepen of the is ee Fsponenia phase ~ Aico tial growth in whlch alls dol wth const cl geeration Tiers nexponentaly zasing el number. Tisoccue int ibe whetere neo few cll tat ress tent dete fo the sed, a wise procs Jo nok {ecumlateto high eels Soca statins phase, = Feed = A process heey se open cr ution of he tps fa system fps (thee) witeton ™ mene Feedboc ili A camvon cot messi Un ie torn hh a pot Ihe at ey nth pa en of one hse ay that pres ht lc. GLOSSARY 267 Feedforward top (HL) — A yotirn with hye es 8, Yeon Zin wh X asa diet oY Mas and¥Yocaneted edgetoZ, Tie 21 stork retin say kgs mat Ia com perf avails ask ch sig active ea. env acelin, an pte pesto 1-tune property — A praprty of billet tameacs ofthe ic apt rus opet Piestonder kinetics — Mathomatce description of the ate of am ezine recto i the Unit thee te uate conentatio sey ow tar fe starating he eyes ha {hese al GK) FS, whee vis the ate or ehizyne, Fie theca conceesn Kathe Michal ons and Ss esate concentration Ss al Mie Meo inten eer Kinet 1 ports sense na the iene ‘Faget ral flagella) — Along ment soe tation dives cess through fad msm, eta ye gl te Panetionationy te scat of setting 39 geass stentless By "tecpngtw els hr sess sheet ral omen ses ona ~ ihe tt ai ufa conse bh ditt ys of ore tn Cre ‘Mone sath une tne sNA, ih the ns ‘Mae yon he gn ely gary DNA sgn ale the rer at MAM Endng ates tr anon acre atest ste ftensrton Gece en a ins hk to sn 9 cf alee hat nest Yo pram & Sania fen. An examples ed lores Gone rount he tn nce by age Stes HERNA teat fay the se, en She nNA vay arom even ine. tine maga od rng Gane code the nayangetnen te fons onthe 20 sme acs The toe ie Gealimpea acre nei dit he taste cane er tne of gn ss 3 lain se to en ‘amplingetes nthe ns coe ner Gomme hel eesti sl ale {Gucre A snyleape sng snes ney etl Mon i) Asn perenom sree offen dtc fhe pce vnc ssencer snd artis ened at etrptnaio Homotgms sini vitseofconmon ein Say nc ore sean A clan Hrd hy esse lar ht a les ei Iman Tes yw the bly pcs il fa gn ti In spn inet whe wd els on pce sue tha eng atk sai, ‘rewonaname Incohee e onan op — eva pin wich tig of he ret peo X40 this oppntean eel ignofiindnct pt om th ¥ 7 Incoerat pater Apa vith ssn he ie wich tee exis pif mesh ern ct ais eee thse ms sah a he oe i a he path Sire " Inte et ee insincere of eeu is men the dlesised wutput) is negatively fed back into the input of the device, Integral feedback can tention orien a ope hy ees cl att as mh ron an rans tc NA. Te eer ancl eet eae h ch ons ges gently shoe‘eases nt aco slate pes (qs ae hh pum tae io teclicant LA, wheoe ft enkin, atime pomp fhe als gc, seer ees stice, pret a phvos eal “nde excise The la pete ‘eps by act a acted CIR. te ebay the DNAs he syn ed lnthe ese culace (latins diate flac a at or no etal Aelita FG actoue = A super ied hy F clas an energy ad carn sours, asng the be gone expres Teo the daca igund — A tei hat special binds the ling tena wet Mathematically cotelled comparison — A conpartinn btwwen melons th i carted Wek equivalence fs many internal and eaters paraetereas pie betyen te ler. rate desig Saragen, 975) Internal parameters inl ahaa parameters sch sh ‘elite of he potins hit Wake up te east alexa poametrs cae esd ‘output ropeten, such a steady stale Membrane — A sirvtute consisting pineal ot ii kc that define the vter bares of Membrane potential — ibe den cis levis pil inside alos fhe cl expres ss ‘age tlie the aside voltae. Mean putts malimalned by prten purnpe ‘hat anspor ies srs the rersbrane a the expense ef ensgy spe by ATP Michucts-MentenKinetis — A rates dseipion the rie ofan seymatic reaction 38 funcliomot the conceteston ifthe eluate Therate sequal tov ESA +9), where wi the ate per enzyme. Fis the etayne concen, ith atte concemraon ad Ke the Miceli constant, When S > K, ome nai revo-orkt kine ete v Eyal when ee ose abn ator bint ate (tk) 15) Micron = Oa slinth ort, Madurity A peoperiy aa systns hh ca b paral nto pr independent su yen Marple — Alek protein th deters patil pater Mnphgers ind pee esp "ooo gg sal tansucton paiva within the cls to be pated. the signing lel th elle sun it el tes crn msg ee Morphology — Piyscalshapeurnl sear ImliNA™~- A nucrooloole wade of sequence of four typeof ss A Gs DTrace {ste acess by whi an RNA-plymerse erye padi a RNA mle thal cor fespotl tothe base sequence on the DNA twee DNA Ts apd ta BNA V) The mRNA {stead hy sbsenen whch pvlices protein asin the MRNA sequence Mutation —A berate ung nde ase yi uous the hearse [Network motif A pallet terse tht revit ina eter many eet Network i cate deleted a tern tstcoreh eaien ha e Mave ctor [Noutom acre cell) ~ Cl specahneb i ese, tas cont geal the heron 5 [Nace A siucture ncn by 9 enn bo "ain the chromosoncs, ‘Operon — A group of genes trancentet on the sic mRNA. Lach ge spate ranted, Ope ‘ons re Town ly prokaryote, Peptide ~ chain of na sch jied together by peptide Hom. roti ae og peptides. Point mation — A chang x single ter (hse palin the DNA. Poison dsieibtion m= A dsribotion that charatciags 9 rdom proves such asthe numbee of Isads ina eins experiment wt any tans, Nand sal probably fread, pe 1 themean numer af ea c= Bt aan ta isn proces feel othe vean. a mad hence estan deviation the squane soot ofthe nen ae Promoter — A relay epi nf DSA hat cont the transept ae ee The poole ‘oni bing ite fe KNA paljmerase GVA the yan hal tansy yet iw easy calle (ttn baer) that a GLOSSARY 263, proce nitNA, ach promoter ao sully cons nding tes or tanssigtion Ft [otcns the transept factor hn let the pty hat RNA i se transcription af an NA, Protease — An ett tha grades rons Prati acon treed for dota ally lt eye ocx any enkyaie poi are age the patron by nym tht sta a chau of wun wl to he gt pro Dire protis an have dierent gration tes Protein — A lang dain of arin aks fon the oro foto hee ain ac] tht can ‘erie ina arctural capacity was aye Fach ei encode ya ne Prins te Profuced in bosomes as on information end on im mRNA tht asc os the gee ‘Protein kinase — An emyne that sachesa poste (PO) gro wa pron ad heey sees bangs Receptor == A eon neil, ously sated inthe membrane of he cel ha seas a pr ie chanical, When the ppp chem he ind nde te Binding eo he weep, opal tanslation coca te agen with the cl Repressim thesbeld — Cancetstion nf ative repress ere for halos repression ofa ‘e Repressor = teaesription Fact ithe promoter a gone Ribosonse— A stroctute in theeytploa nde of abot 100 yrusing and pei RNA moles hat sive thee of pradton of tere trated om oRINA he ese, amino tide are asm to form he protein ean acc oa er speed bythe cds tov ahe m}'NA. The anion eke te Bra in the rbowone by ¢RNA accel, she ‘eal the RNA cde Fach H0NA fees won ein ail ined tothe tae lusess thea rameriplon when it binds a specie te roca [RNA Poth merase (RNA) — A conpx svi sins th fom an zy that esas ON Tigo RNA. bust operty~ Property abst wth espe to parameter 0 nscasine changes in ovametery, ‘Sensitivity (Parsusersensivty) "the parame ensivy solisin of property X wil epee topparamter Yap asi edie changin Ys dog ye cx ype eB OR Se ing AF Semary transcription networks — Transcription mek tht resp to eneanrera ad ater Sul ng ich emanates, a Ted to cages gee expression. These Ret ‘evi nstto fet rapid anelywhin les tha ell gnerto tine ar uty Stationary phase A sate ba which cle cease to divide od grimy hat ocean revel con Tins are avfooras ach os when the bacteria ron out of am eee] uti See ao ‘expaneitl phase “Teletogy The we of sgn on purpose a exlanatin of matual phenomena Teamiription factor — A plc Tha cela the tranerpion rte of spc target genes. ean expo tors usually hve to mole sate, ctive a etn. They east betwee these state a rapid tnescae (eg microsecond When stint eanscipin itor Ios spose sites the DNA to at the ito eassripion ition of gel ees. ‘Also calle transcrip ego, Se atest represen, Tramserption stork — he st tansriton strata cel Tevet is mae of nodes Tied by Snect ages, tach nde reprsete seem im betel, an operon Fach ege270m GLOSSARY fa tanscptona neato, XY miso tht the praia enc gene X ta rion ana onscreen gene. we OR gate (ulusive Ot) 4 log aneion a tw iat at putea one Wether, bt et bth Tats veal to ve Yeast — A singe elledcabaryote unica angus this book sally the baling ys Sa ayers orev Yess is saed fe brewing and ead maki an 9 wl esearch adel organi. {erw-order Kinetics ~ Matbesmtial decription ofthe rte uf an eneymatc resto the Ht ‘ehere the abate concenition i eturstng eich tha the rate eo tow ky were the ate perensyineand Kite ensyebe cncentuin. 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Lege Andrew, He sey, ‘€ Toco and EP Roth. 2005, Metis, heme and thematic nape of an negened Sacha mmyser cerevisiae interaction netwook J Bil 46 ‘pha, VF Myulinaos, Adana, ad 1, Lipson, 2005, Robots selGsepmadacng aahines ature 45: 163-164,Index A Aspesion, 138 Age. 12 Ame 45,18 Arabian ten, 545, 2.2 gine nya 26,77 79,80, 218-29) Autres 274,100 acostins n proton ae, response 31-34 ates, B aii i 103 actrees, 196-1 ne Laer moe, 6-18 Nas expreson eel 15,207 olan hg ig in, 28-299 stb, 79, 22 c ‘Gaoorbab ent 98118 125,260 {AMF 17, 54,64, 206,215 aaleaton 186 acide sdeunage a rprecrreanade, 9,14 In developmental anerpian nto, ie 268 sy. ea erat ine 2, 3.78 97 102 18 ecg proces 17 herta 99| horas Iota 3196 ection of sal gion, 36 act api, 8 fetal int Angel dha 8 resol 8-150 on cit, 0 horas, 229,264 ‘radun clock. 117 ut patos andes Chest eet 259 oon, 17191 Caren FPL 47 Goin dsteton, 125,127.18. Cami lek op, 77,18 Constative expen, 20 ined tnt 89 Goaperaii. 245. {ost beni ay 194,198,208, 207 Chose tt,21 D ales oe 12 Degen sce 113 rooyen, 63 respons ise a, 20,3464 25sel omban Hl 15178287 gue mses don nt orks). 6 wei ests (ORS. 75,4588 9, tls 9 aseptic sta, 3,909, Seeds eet, Disoctto conan 242 WO sean sping segue We ge: peserving ton nt as, al aarp to 2 n Agiering 27 tal eo 16,297 Il ese 9 iis a sds i288, 259 reba 238 sag tai aye ne destondly,1 ener e207 gunn ining ‘pa cyan i HB twas. 177 ey 29 HR else Bk Rey mel ve a 216,281 vor pre ing tte, 228 ror 9 ari co 9.20, 25,2. 50, 36, henna pte cet of, 190 Alan in 27 98.223,224 24 spot tein 8 sat asp, 17, 19 32.145 46189 aca say 222 r Fimo 28 Fea ahi, Felice sama so eto, ko segulation Sampo scion 8 nso. 16 coe 7.8| Pe nepali, 99 18, 304 boat "uch of amp nate logs, 92 Peas emad ap (YL) #88208, (C1 97, 30,52, 50,103 eon 99.6.8 9,125,206 coincidence detest 125127 eget vein 68,69 ete oa 20 TT 87 562,63, 6,65 108, Ince, 57.68, 98-9989 ap, 122, 124, 1 239 aon 75, 81, 83,7, 106238, posse det $8, 53.70,125 os ule yen 57, 58,62, 70,22) response acelean 6,70, 293 2p sentve dy 50, 52,54.90 oy tte giao? ee ae 2 peso 14.48 65,70, 98-99 anit inet sone 87 nto et 59 HIRO se tensor sae ee 16 ncn pens, st i st ut (FF 8, 88,87 Festondrlier age 245 Pvc aac, 191196177 uate outa, 22-209 Hog, 86, 4386,257 re go 199 ray patersing 15672 G one) hasan input foto, 207 ov demand 26 system denn 27 ie eet deya, opto, 193-24 ‘viene section of ews ase woe nt 08-207 optinal epron elof patina essa contin 94-200 eel fe prot, 195-196 oso Lac protein, 196-197 fies Fri an optimal expression eel 17-199 pn eistion in vtable environments, 21-205 ‘Gene elton eran leo, 215-254 fern role fr muliegulstor sytem, peu rule for gene epson ad om main rior 20-222 Sevagea dena rile, 20 Gene code 15,176.17. 391 G1 ee Gren once posi hab epson 77.258 Greta dea tn, 19 INDEX 297, vest putt 9, 28, 36.6, (rot as, " en Ul oti 18,3927 ani 32 295, Misia, 222-225, 280 Hoos ts omlagi rs 68 } et PEL 5.65 fo-degreedatebation. 258 Ince tno, 224 Inpt fonction, 13 Inge otc, 0,186 185,156 297 Ingenta ee mode 133 age K inti posed, 75-191 vei recog ese in cl vara In DA epi 87 in proven degradation, 17 recagpliogselfand ns by i L Iaborataty vc aperient, 1 ae syste 1, 14,204, 23,2 se poe. 185,196,224 ein esto (FO), 7879.81 Leainess, 8 207 Oe atin tout pans. oe lackeonmashaniam, 99ag mp ct, 85 Dae doin ete 216 217 M AP one ele, 106 ALL Maly emt ems, 38, 3 ety tio, 2 Mir chat Meno uate, 16), 20-252 Mon angers Wyte soe, 207 Mosel) ort eer, 18380 fe toe, 13 re ap 89 Ising 247 Moby, 2,20¢ fenced promt 1 quate reson 9 259 spatanconsenton 9296-236 Muephege19 Aegean, cena 16,165 ech ag ie 159 aerting dl 9 raises of ei, 6b Mut ee aa Net it ii desi pat orton, 253-258 voor preps 96,6 laeinton 118 etralation, 13 rela degrada, DS Fm nese net 125 In sgling ter, 05,127 Mult utp FF 8,83 86,82 N Netw eye epee a 46 event anion, 97 spent cin canes 89,258 duly qpantitatne me 259 en sani 7 imi 89 seosy tampon 57.8 ula spare 48.297 Nevworh nti 2, 27 69,205,233, pote 9 1% 27 FPLanas Infrmatinn proven Santon A sccorteeeot dsl ignad eam oro sets, oa soompi i S197 Neen i Aerlopmenia transcription nwo, sero ed oral atic ovat crt 12-104 long etsetion ces sd owkepment i. 2 sequin fibavk ya fess, 2 eae pao Gack ops fi cso making. 95-10 lnfnmation rocesng seg eager pereptrns 106-115 nl ae peers prorming ‘eta computa, U-215 Amin th eon setworke ean 6 uknp n mearna metoks pes ger perceptron in the Clune sia et 25-36 stra eames e104 15 Nese aetnorks aris. 186 Ness eth 38 eal Network nt Jn denlpental sign ranstion, sd evo networhs Highs aopsin, 16 network 126 Noise el el abit, M3752 185 ‘raed, 290 eat storgaion an 262 pontveatozeglaton an 368 oO (ne dine dfoson, 202 ON peliss prior dae for, 32 opsoms psi an eli, 191 Ostdgee santos 258 > Pavater sesvy. eSemiey este to, for ON ules, $2 age lan, 63, 100 Poop, 107| portance ia sing, 183 onporpaton 106-108, eee expat dynamics 58 Pov coma 2 Prorat atachmest, 258 Prodctiodegeadstion equation 32 Promote 88,289 Proce) leaden, 19.07 nse coca, 15,106 10%, 130 ins enor gral BD aed, 195,196 yi erations, INDEX 299 oO an sig ® Rendon ewer, 27 Ratchet newer 91 eset, 18, Rosana 1 Relat thick, 12 Repellents 196,102 pesmi 18, 243 Repos. 8244 eascade,94 202 seat of 47,62 64 degen nd 20,3404 FF 61.82 ts 21,62 In axgtvessorgetion,3155 postive aotonegltion, 37 ini ove ec 2181 ramcriptos aterctins 27 Reser egnceciag, 299 eso 68662 15,287 NAR sce BNA puerto RNA pease (RNAD, 8 66 acter eet 135187 efoion of 135 eats atoaguaionand af erporl programs 4 Robust peri ndevopeent, 199-178 expen morphsn paler ae sot ribs 61-162 ncaa sbesteay seeance _noephoge dren, 16-165sec nt that poe dri Kelas rb ping. sins pn datagsishigbtcen etnias reserve Sage dea 217,208 sy tons 89 se 30 Sma eatin, 1 16 165,287 Sens. 38,1, 1 196 Seosoey tans simak, 98 Seyoetion a times 22,9, 98, 29,294 Seria dilation, 19 Siglo tuk md combina cata, 88-89 Sign eansduct ostsks, Use aio eaworh tsi denopoena al Sito tamadction apd a Sevlpmenta tantra meio i et aor persons in 6 trk tin Sg sented 52 SIM ze Sigh ie Simpy 1, 268 88233 Single inp alae (175,76 ‘tof 0-8 empuel programe and, 7-80 Spars tons 8,257 Spelt 10 Stay, 16,285,282 Se pete, 21 Step fet S| Subgept a2 Sybil 6a Sythe gece Spt biloy 1 Tet recep 9, exopitn. intic rte Tempra pregrans ef expeson 7.29 bse we, 1 “hes of eet 0 “Te evi 8,27 Topol generals of dan ota a3.122 foto 7 Inert sponse tie 2? Teasers tvs R248 eascron nator 828 itive problem fl, 5-7 eveopsnt 9791.97, 98127 Aynanics and repose ine, 1-22 Eesha sleet tancrcion swaths 7-18 ests and een, 2 Ingato, 1345 Toe pt Fons, 3518 oslo 16 spans a eta 9-12 hater cote 299 egress, 297-299 ie nesue of modular, 259-260 ‘ep proprams ad bs srctne of, win 80 enon eration, 189 Sato poses, err ae, 187 u bigs, kei proeing and 7 nena fico pattern. 14 nari input sous 87 v Yorba Note y Me 61248, 48, 57, 4,89. 9, 91,16 128, 135, Zz Yeo-ntar ati, 3,251 Tender west, 156157 DEN,