GeneRegulation.

pages Gene Regulation Notes

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1. Cybernetics (steersman, governor, pilot or rudder) (Norbert Wiener and others) Governor and relief valve on James Watts steam engines Problems with overshoot and oscillation. Systems and Controls on Car: air=f(fuel, temp)=f(load)=f(speed, incline, accel., gear) Brakes, emergency brakes power steering AC system cooling system exhaust system engine-generator-battery-capacitor-distributer-spark more electrical: horn, lights,fan, etc 5+ general solutions: Natural aspiration /carberator Turbocharged Fuel injected Hybrid gasoline/electrical (Prius) Electrical Vocabulary and symbology. Effect of one thing on another may be positive, negative or neutral. Positive regulation -> Negative regulation -| Simple Cascade: A -> B -> C -> D Double Negative: A -| B -| C indicates A -> C (May be unknown intermediate steps) Negative Feedback loop Positive Feedback loop Positive Feedback loop A -> B -> C -| A A -> B -> C -> A A -> B -| C -| A or A -| A or A -> A

(double negative is same as a positive) Q How can you maintain constant levels of a protein (homeostasis)? Q Draw Graph of Cascade, A,B,C and D as function of t. Q How modify the Simple Cascade so A is followed by B (w/o A) and so on?

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2. Differential equations and dynamics. Let x be the concentration inside the cell of your favorite protein, X. Then, the rate of change of in the concentration of your protein is given by the summation of various rates: dx/dt = synthesis rate - degradation rate - dilution rate dx/dt = syn - deg*x - dil*x

if dx/dt = 0 (meaning that concentration of X is not changing, is constant) then the steady state concentration of X is: dx/dt = 0 = syn - deg*xss xss = syn/(deg + dil) All else being equal, the steady state concentration of X in the cell could be increased by: 1) increasing synthesis rate 2) decreasing degradation rate 3) decreasing the dilution or growth rate Similarly, the steady state concentration of X could by decreased by: 1) 2) 3) Note: Growth, dilution, and degradation are all exponential functions. Given growth equation: N(t)=N0ekt - dil*xss

Derive differential growth equation dN/dt=k*N Show dilution rate = growth rate = ln2/doubling time

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3. Some Possible Points of Regulation (almost anywhere) To regulate level of a protein we might regulate at a variety of levels, in a variety of ways: gene-level: increase/decrease gene increase/decrease gene increase/decrease gene increase/decrease gene genome rearrangementspop-outs. copy number modifications, eg methylation accessibility, eg chromatin structure structure or activity, eg supercoil deletions, duplications, inversions, and

transcriptional: increase/decrease rate of initiation (eg initiation by RNAP) increase/decrease rate of elongation (eg pausing of RNAP) increase/decrease rate of termination (big effects downstream) post transcriptional: increase/decrease rate of modification (eg splicing, editing, polyadenylation, 5 cap, transport, etc) increase/decrease rate of degradation translational: increase/decrease rate of translation (eg RBS structures) increase/decrease rate of elongation (eg translational pausing) increase/decrease rate of termination (could limit overall rate) post translational: increase/decrease rate of folding or refolding (chaperones) increase/decrease rate of degradation (eg proteosome, proteases) increase/decrease activity by binding ligand post translational modifications increase/decrease activity by modification of protein, eg: specific proteolytic cleavage of zymogen or precursor acetylation, methylation, phosphorylation ubiquitination (ubiquitylation) adenylylation (AMPylation) gycosylation, farnesylation http://en.wikipedia.org/wiki/Posttranslational_modification Q: Distinguish between global regulation and specific regulation.

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4.Dual Regulation of Lac operon in E. coli

PI

lacI

Plac

lacZ

lacY

lacA

PI is a promoter for lacI gene. Plac is a promoter (binding site for RNAP) for lacZYA t is a terminator (RBS is not indicated) o is the binding site or operator for LacI c is the binding site for CAP (catabolite activator protein) lacI is an ORF that encodes LacI, aka the lac repressor. (An ORF is an open reading frame than can encode a protein). In the absence of allo-lactose, lac repressor binds the operator and thereby hinders binding of RNAP to the adjacent promoter Plac. lacZ is an ORF that encodes LacZ, aka beta-galactosidase. Beta-galactosidase cleaves lactose to yield galactose & glucose. As a side reaction, it also produces allo-lactose. lacY is an ORF that encodes LacY, aka beta-galactoside permease, aka Lactose Permease (symporter) lacA is an ORF that encodes beta-galactoside transacetylase (transfers acetyl group from acetyl-CoA to beta-galactosides) (exact purpose in this context is not well understood) CAP (catabolite activator protein) binds cAMP, binds a specific site labeled c and recruits RNAP. Adenylate cyclase converts ATP to cAMP (3-5-cyclic adenosine monophosphate) which is an intracellular molecular signal. allo-lactose and IPTG are inducers that induce expression of the operon by relieving repression.

PI

lacI

Plac

lacZ

lacY

lacA

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Transcription is lowest in the presence of glucose and in the absence of lactose. In the presence of glucose, the preferred sugar, the transport of glucose inhibits adenylate cyclase, cAMP is low, and CAP protein is inactive and does not bind its site c. In the absence of Lactose, the Lac Repressor binds the operator (o) and interferes with the ability of RNAP to bind the adjacent promoter (Plac). Thus, the activator is off and the repressor ison the DNA. Lets call this minimal or basal level of transcription as 1x. In absence of glucose, adenylate cyclase produces cAMP. cAMP is bound by CAP (Catabolite activator protein) cAMP-CAP binds the CAP DNA binding site (indicated by c) cAMP-CAP-DNA can recruit RNAP to the adjacent Promoter (Plac) and thus increases the rate of initiation of transcription. Maybe 20x. But, transcription rate is still not optimal because: LacI (aka Lac Repressor) is still frequently bound to the operator (o). Binding of LacI to the operator prevents binding of RNAP to the adjacent promoter (Plac). Thus, cAMP-CAP-DNA can only recruit RNAP in the rare moments when the operator is not occupied by Lac repressor. If lactose (aka galactose-beta(1->4)-glucose) is present, it is transported into the cell by Lac permease and then cleaved by beta-galactosidase to yield glucose and galactose. An occasional side reaction produces allo-lactose (aka galactose-beta-(1->6)glucose). Allo-lactose binds LacI and slightly alters its conformation so that it does not bind the operator efficiently. Alternatively, a synthetic compound, IPTG, can bind LacI and similarly interfere with DNA binding. By itself, IPTG or allo-lactose increases transcription by, say, ~50x. By itself, cAMP-CAP, has ~20x effect on transcription. Together, the combined effect is ~20x*~50x = ~1000x Medium glucose, no no glucose, no glucose, no glucose, no PI lacI lactose: lactose: lactose: lactose: t c Effect 1x 20x 50x 1000x Plac o Bound Proteins LacI (>>RNAP) cAMP-CAP LacI (>RNAP) RNAP (or not) cAMP-CAP-RNAP (>>LacI) lacZ lacY lacA t

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5.DNA Looping and the lac operon---action at a distance

OI

OZ

effect of removing additional operators. effect of altering spacing between two operators. effect of assisting in bend formation between two sites. each operator site is bound by LacI dimer. DNA between sites can loop to allow tetrameric interaction between 2 dimers. This cooperative tetrameric interaction increases DNA-binding, half-life of complex, strength of repression. Mossing, M. C., and M. T. Record, Jr. 1986. Upstream operators enhance repression of the lac promoter. Science 233:889-892. Effects of DNA bending proteins (IHF) in other systems. At what level or process does LacI regulated gene expression? Is LacI a positive or negative regulator? At what level or process does CAP regulate gene expression? Is CAP a positive or negative regulator? Is IPTG an inducer or a co-repressor or a co-activator? Is cAMP an inducer or a co-repressor or an co-activator? Can an activator also be a repressor?

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6. Cooperativity, kinetics and binding curves. n=hill constant = cooperativity factor = order of rxn in [P] Protein-DNA Complex --> Protein + DNA Site [P] is concentration protein, [S] is concentration of DNA Site. Analyze in terms of Free DNA, Bound DNA, or Fraction Bound. (But math is similar for protein-nucleic acid, protein-protein, protein-ligand & nucleic acid-nucleic acid interactions.) Bound/Free=B/F Kd = [P]n[S]/[PS] Kd[PS]/[S] = [P]n Rearrange Take log of both sides substitute B/F = [PS]/[S]

logKd + log([PS]/[S]) = nlog[P] logKd + log(B/F) = nlog[P]

Q. Draw log (B/F) vs nlog[P] for n=1, 2, 4. Fraction Bound = Y Y = [PS]/([PS]+[S]) Y = 1/(1 + [S]/[PS]) divide top & bottom by [PS] multiply top & bottom by [P]n. substitute with Kd

Y = [P]n/([P]n + [P]n[S]/[PS]) Y = [P]n/([P]n + Kd)

Does this expression look familiar?

Q. Draw Y vs [P] for n=1, 2, 4. At what [P] is Y=0.5? Q. What is the relationship between Y and B/F ? Y = [PS]/([PS]+[S]) What next?

Q. Scenarios 1. Monomer binds DNA. What is n? 2. Lots of monomer, some dimer, dimer binds DNA. What is n? 3. Little monomer, lots of dimer, dimer binds DNA. What is n? 4. At beginner of binding curve, mostly monomer, at end, mostly dimer. Dimer binds DNA. Sketch binding curve.

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7. Cooperativity and Thermodynamics Two different proteins bind to two adjacent sites on a piece of DNA. Potentially, the binding of one protein can either help hurt or have no effect on the binding of the second protein. We say that binding of the two proteins is, respectively, positively cooperative (or just cooperative) negatively cooperative (in the extreme, mutually exclusive) independent (or noncooperative) General Free energy equation: Gbinding = G + lnQ = 0 = G + RTlnKA Energies Add. rxn 1, rxn 2, rxn12 (both) G12=G1 + G2 +Ginteraction Ginteraction can be: negative (positively cooperative) positive (negatively cooperative) zero (independent) Allostery=other structure. Ligand binding can shift equilibrium between alternative structures and/or trap structures. Str1 + Ligand <--> Str2 + Ligand ---> Str2-Ligand Complex. Analyze reaction in terms of coupling and free energy. If free energy of binding ligand is worth 11 kcal/mol, how much can Ligand binding shift equilibrium between Structure 1 and Structure 2? Are there other ways to draw this? Other possible complexes? = G - RTlnKD

GeneRegulation.pages 8. Trp Operon of E. coli

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trpR

Ptrp

trpL

a t a

trpE trpD trpC trpB trpA

Trp Repressor trpE, D, C, B and A encode enzymes involved in the biosynthesis of the amino acid tryptophan, aka Trp aka W. Logic: When Trp is low, biosynthetic genes should be expressed, in order to correct the lack of Trp. When Trp is high, expression of Trp biosynthetic genes is superfluous and and thus an inefficient allocation of resources. trpR encodes the Trp Repressor protein, which binds the corepressor, Tryptophan aka Trp aka W The TrpR-W repressor-corepressor complex corresponding trp operator (indicated by with the binding of RNAP to the adjacent thus reduces initiation of transcription binds to the the o) and interferes trp Promoter (Ptrp) and for the operon.

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9. Trp Attenuation (of termination) P trpR Ptrp o trpL a t a trpE trpD trpC trpB trpA t

the letter a, t, and a represent segments of DNA (and of the corresponding RNA). Segments a and a are similar and both are complimentary to segment t. Thus, the RNA can form two alternative hairpin strs, at or, alternatively, ta The second structure, ta is a terminator that induces RNAP to terminate transcription. The alternative structure, at is an antiterminator that does not terminate transcription. By failing to terminate early, the RNAP is committed to transcribe the structural genes trpEDCBA before reaching the next termination signal. In E. coli, translation typically begins long before the mRNA is complete. Thus, the ribosome loads onto the message and chases RNAP. This coupling of transcription and translation offers a unique opportunity for translation and transcription to directly influence one another. trpL encodes a small leader peptide whose most distinctive feature is a tandem pair of UGG Trp codons. If Trp-tRNA is plentiful, the Ribosome translates the leader peptide, melts the at antiterminator, facilitates the formation of the alternative ta terminator structure, and thus promotes the termination of transcription by RNAP. Alternatively, if Trp-tRNA is rare, the ribosome stalls or pauses at the tandem Trp codons. The at anterminator structure is thus not melted within the critical temporal window and RNAP proceeds onwards to transcribe the structural genes trpEDCBA that encode enzymes involved in tryptophan biosynthesis. This, in turn, leads to more synthesis of tryptophan, generation of more Trp-tRNA, and thus to a reversal of the regulatory state (eg, increased repression by TrpR and to increased termination by ta.

GeneRegulation.pages SIGNAL INTEGRATION

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Logical and Boolean operators: AND OR NOT BUTNOT IF IFF THEN IFF (A AND B) THEN C IFF (lactose AND NOT glucose) THEN strong expression of lac operon.

SIGNAL TRANSDUCTION (especially across a membrane) signal is membrane permeable, receptor is inside cell. eg IPTG. Signal is transported (eg glucose, lactose) 2nd or intermediate signal is generated (eg cAMP, allo-lactose) Signal stays outside membrane, but is sensed inside membrane. How can this be done? ex Couple binding of Ligand to state of a (dimeric) membrane bound receptor. monomer - dimer equilibrium rotation of units in dimer relative orientation of monomers in dimer (push-pin) E. coli: Lac, SOS, Trp, B. subtilis: SAM, Com, Spo sigmas Vibrio Fisheri: lux?