DNA

September 26, 2012 10:04

Nucleic Acids
Nucleic acids = polymers (RNA, DNA) Monomer is nucleotide Phosphate group Five-carbon sugar (ribose/deoxyribose), carbons numbered from 1' where base is Single/double ring of carbon & nitrogen atoms, nitrogenous base Pyrimidines (single ring): Cytosine (C) Uracil (U) Thymine (T) Purines (double ring): Guanine (G) Adenine (A) RNA is made up of ribonucleotide monomers DNA deoxyribonucleotide monomers Structure of chain

Sugar-phosphate backbone is directional: 5' end (3' carbon unlinked), 3' end (3' C unlinked) Nucleotides added to 3' end when polymerizing Written in the 5' -> 3' direction

Genetic material
Must contain information for entire organism Must be accurately copied Should account for known variation within, without species History: 1920s1940s: expected protein part of chromosomes to be genetic material
Lecture Notes Page 14.1

 1914: fuchsin dye stained DNA 1920s: DNA was in chromosomes (right place, varied among species, present in right amount), possible evidence for being genetic material Avery, MacLeod, McCarty (1944): hypothesized that purified macromolecule (which is genetic material) from type S bacteria (the deadly one with capsules) could convert type R to type S Demonstrated transformation of bacteria from DNA Hershey, Chase (1952): tested whether protein or DNA in bacteriophage was responsible for genetic material

Measured where radioactivity was; experiment 1: radioactive phosphorus was in pellet, experiment 2: radioactive sulphur was in supernatant Chargaff's Rule: in double-stranded DNA, # A = # T, # C = # G Rosalind Franklin: determined helical structure of DNA via X-ray crystallography Crick, Watson, Wilkins, Franklin

Lecture Notes Page 14.2

Nucleic acids (continued)

September 28, 2012 10:00

DNA Structure
Strands antiparallel in double helix Hydrophilic sugar-phosphate backbone faces exterior Nitrogenous base pairs face interior Two different sized grooves in helix (i.e. not symmetric) Major groove Minor groove Purines <=pair with=> pyrimidines A-T 2 H-bonds C-G 3 H-bonds Binding sites on C=O groups and N groups Stabilized by hydrophobic interactions in interior as well as H-bonding between complementary base pairs Base pairs are exposed in grooves

RNA
Also sugar-phosphate backbone, four nitrogenous bases Differs from DNA in 2 ways: 1. Uracil (U) instead of thymine (T) 2. Ribose instead of deoxyribose Presence of additional OH means RNA is less reactive, less stable Can be a catalytic molecule; ribozymes are enzyme-like RNAs Theory: early life originated with RNA Full genetic material Unlike DNA, can function like a protein Secondary structure of RNA Complementary base pairing Typically forms H-bonds between bases on the same strand Often observe hairpin single-stranded RNA

Lecture Notes Page 15.1

Replication
September 28, 2012 10:30

Replication
"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." - Watson & Crick Each strand of DNA has all info needed for copying

Lecture Notes Page 16.1

What is the mechanism?

DNA polymerase catalyzes replication Demonstrated by Kornberg (1956), who found DNA polymerase I Part of large replication complex Unidirectional polymerization: each base on the template strand gains a dNTP (deoxyribonucleoside triphosphate) (the form of a free base) All chromosomes have origin of replication (ori) Replication complex binds to ori Replication occurs in both directions from ori, forming two replication forks

Lecture Notes Page 16.2

Note that these diagrams only show leading strands and not lagging strands. To be continued

Lecture Notes Page 16.3

Replication (continued)
October 3, 2012 10:00

In closed circular chromosomes (prokaryotes)

Single ori 15 million bp in chromosome DNA polymerases very fast; in E. coli, can reach 1000 bp/s Type II topoisomerases cut both strands in a duplex simultaneously Second duplex (i.e. new DNA) slips through the cut Cut is re-ligated

In linear chromosomes (eukaryotes)

Larger chromosomes, about 80 million bp Total human genome = 3.3 billion bp DNA polymerases much slower; in humans, about 50 bp/s Hundreds of ori in humans to increase speed of replication

Leading strand synthesis

Leading strand = toward replication fork 1. Helicase uses ATP to separate strands

2. Single-strand DNA-binding proteins (SSBPs) attach to separated strands to prevent closing

3. Unwinding creates tension down the helix, so topoisomerase cuts one strand then rejoins strands downstream to relieve this tension

4. DNA polymerase requires primera few nucleotides bonded to template strand with a free 3' OH group. Primase (RNA polymerase), synthesizes short RNA segment that serves as primer.

Lecture Notes Page 17.1

 Remember that new bases are always added to 3' 5. Synthesis of leading strand begins, moving toward replication fork

Energy from energy of dNTPs In E. coli, 5 different DNA polymerases; responsible enzyme is DNA polymerase III. In humans, 14 different DNA polymerases (some for error correction); chromosomal DNA replication is DNA polymerase

Lagging strand synthesis

Synthesized discontinuously in direction away from replication fork; the fork moves forward and creates gaps 5'->3' directionality preserved Primase-formed RNA primer also required Discontinuous fragments of newly synthesized DNA are called Okazaki fragments

1. Primase synthesizes RNA primer

Lecture Notes Page 17.2

2. DNA polymerase III synthesizes first fragment, then reaches primer and stops

3. Process repeats for multiple Okazaki fragments 4. DNA polymerase I removes primer, replaces with deoxyribonucleotides Two enzymatic activities: Exonuclease (removes primers) DNA polymerase Uses 3' end of next Okazaki fragment as primer for new dNTPs

Leaves gap between former RNA primer and the Okazaki fragment 5. DNA ligase closes the gap in the sugar-phosphate backbone

Most of these enzymes around the replication fork are probably in one large multi-enzyme machine: replisome (replication complex)

Lecture Notes Page 17.3

Telomeres
Region at end of linear chromosomes = telomere Leading strand synthesis is normal, but telomere on lagging strand shortens during DNA replication RNA primer cannot be replaced with DNA because there's no free 3' end for DNA polymerase to work Humans: ~2500 repetitions Chromosomes lose 50200 bp with each replication Usually cell dies after 2030 generations

Solution Telomeres are non-encoding genes; repetitive bases TTA GGG Telomerase in some actively dividing cells (bone marrow stem cells, etc) adds more repeating bases Has its own RNA template Doesn't have to "read" any DNA or copy any DNA Functions like a polymerase, adds bases to 3' end of lagging strand 90% of human cancers also produce it

Lecture Notes Page 17.4

Lecture Notes Page 17.5

Central Dogma
October 5, 2012 10:00

Codons
Three-letter codons of bases code for amino acids Start codon is AUG which also codes for methionine Stop codon is UGA Tricky problem: the reading frame affects the meaning of codons! So how does this all figure in everything?

Central Dogma: DNA --transcription--> RNA --translation--> protein

Lecture Notes Page 18.1

Transcription
November 3, 2012 21:28

RNA polymerase synthesizes RNA by transcribing only the template strand Other strand is coding strand, which matches sequence of mRNA (except uracil-thymine substitution) Genes could be located on either strand over a chromosome; affects direction of gene promoter->terminator relative to the chromosome New bases are always added to RNA strand in 5'->3' direction Template strand is read 3'->5' No primer required!

Lecture Notes Page 19.1

Transcription process in prokaryotes

1. Initiation phase: the sigma protein subunit must first bind to core RNA polymerase to form a holoenzyme Sigma factor responsible for recognition of promoter Specific parts of the promoter in bacteria have special significance: -10 box is TATAAT, -35 box is TTGACA Sigma binds to -10 and -35 boxes Different sigma proteins allow recognition of different promoters 2. Initation phase continued: sigma opens double helix and threads template strand through active site ("open complex")

Lecture Notes Page 19.2

 Sigma dissociates from core enzyme once initiation is done 3. Elongation phase: RNA polymerase moves along DNA, synthesizes RNA 5'->3' 4. Termination phase: encounters termination signal in template Can cause mRNA to form hairpin secondary structure, separates transcript from polymerase

Transcription process in eukaryotes

Similar to prokaryotes, but each step has more proteins RNA polymerase II: transcribes mRNA (or pre-mRNA) Requires 5 factors instead of just sigma RNA polymerase I, III: transcribe non-structural genes for rRNA, tRNA RNA modified after transcription (pre-mRNA -> mRNA)

RNA modification 1: splicing Happens inside nucleus Introns are non-coding, interrupt genes, must be removed by splicing Small nuclear ribonucleoproteins (snRNPs) form spliceosome, contain both amino and nucleic acids Exons are coding regions, part of final mRNA product RNA modification 2: capping 5' cap, 7-methylguanosine Needed for exit of mRNA from nucleus,
Lecture Notes Page 19.3

RNA modification 2: capping 5' cap, 7-methylguanosine Needed for exit of mRNA from nucleus, binding to ribosome for translation RNA modification 3: tailing 3' tail, 100200 adenine nucleotides (AAAAAAAAAAAA) Also needed for export from nucleus Increases stability, lifespan To remove all mRNA from cell, use DNA sequence with a ton of Ts attached to magnetic beads

Regulation: Operons
Operon: cluster of genes under transcriptional control of one promoter Negative control: repressors affecting gene transcription Repressors can affect RNA polymerase binding, or block it In lac, lac repressor binds to operator, which is after the promoter; can block polymerase Positive control: inducing or increasing gene transcription Activators

Lecture Notes Page 19.4

Translation
October 10, 2012 10:00

mRNA is converted to a protein, catalyzed by ribosomes

Translation process in bacteria

Transcription & translation occur simultaneously in simultaneously Ribosomes can start translating mRNA before transcription is done Multiple ribosomes attached to mRNA form polyribosome

Starts at 5' end of RNA; first synthesizes amino terminus (N-terminus)

Translation process in eukaryotes

Transcription & translation separated Pre-mRNAs in nucleus Mature mRNAs in cytoplasm

Lecture Notes Page 20.1

Codon -> amino acid translation

Francis Crick: adapter molecule holds amino acids in place, interacts directly & specifically with codon; transfer RNA (tRNA)

Aminoacyl tRNA synthetases charge tRNA by catalyzing addition of AAs to tRNAs Must be highly accurate, high fidelity Use ATP to link amino acid

Lecture Notes Page 20.2

 tRNA covalently linked to corresponding AA is aminoacyl tRNA Anticodon on tRNA makes contact with codon (recognition) Wobble hypothesis 61 codons, 40 tRNAs in most cells In some cases, anticodon can bind successfully to codon whose third position requires nonstandard base pairing (e.g. serine UC_) Only requires precise pairing of first two bases

Ribosome structure

Contain protein & ribosomal RNA (rRNA) Two subunits: small & large 1. Small subunit: holds mRNA in place 2. Large subunit: peptide bonds form They can separate, and come together during initiation

General translation process

1. Initiation phase: AUG start codon Careful! AUG isn't necessarily the first codon in the mRNA mRNA in eukaryotes can contain 5' untranslated region (UTR) Bacteria have a preceding ribosome binding site complementary to a part of small subunit; base pairing! One mechanism for managing abundance of proteins can be adjusted by imperfection of ribosome binding site Interaction between small subunit and mRNA mediated by initiation factors

2. Elongation phase: initiator tRNA (f-Met) in P site Charged tRNAs enter A site, different aminoacyl-tRNAs enter but the one with the right anticodon stays

Lecture Notes Page 20.3

 Peptide bonds form between amino acids on the tRNAs; added to C-terminus of previous amino acid Elongation factors move mRNA down 3 nucleotides at a time = translocation tRNAs move A->P->E, if E site already had a tRNA, it is ejected; A is empty and then this cycle continues

3. Termination phase: when A site encounters stop codon Release factor protein enters site (not tRNA), no amino acid carried but shape resembles tRNA Catalyze hydrolysis of tRNA in P site with polypeptide Ribosome subunits separate

Lecture Notes Page 20.4

Gene Expression in Eukaryotes

October 15, 2012 10:00

Various points of control affect gene expression (and modulate the level of gene expression). (see right) Translational control and protein degradation often have faster effects than control within the nucleus

DNA compacting
DNA wrapped around proteins to create a protein-DNA complex, chromatin Chromatin has a regular structure, several levels of organization 1. Nucleosomes are beadlike structures negatively-charged DNA wrapped twice around eight positive-charged histone proteins histone H1 maintains structure of each nucleosome linker DNA between nucleosomes 2. Nucleosomes together create 30-nm fibre 3. Fibres form even more complex protein scaffold 4. Everything condenses to chromosome Opening up chromatin Chromatin must be relaxed/decondensed for transcription Condensed chromatin -> open chromatin DNase degrades open chromatin to fragments but leaves condensed chromatin intact Two types of proteins 1. Chromatin-remodeling complexes reshape chromatin Use ATP 2. Other enzymes catalyze acetylation and methylation of histones Acetylation (negatively-charged groups attached to positively-charged lysines) reduces positive charge; associated with activation histone acetyl transferases (HATs) histone deacetylases (HDACs) Methylation ~ activation or inactivation Histone code hypothesis: chemical modifications of histones contain information influencing gene expression Daughter cells inherit patterns An example of epigenetic inheritance; not due to differences in gene sequences

Transcription control
Promoters etc similar to prokaryotes Also have gene-unique promoter-proximal element

Lecture Notes Page 21.1

 Also elements far from promoter; DNA looping etc allow them to have effects even though they are far Regulatory sequences that affect gene transcription Enhancers (positive control) upstream/downstream/within introns Silencers (negative control) shut down transcription

Lecture Notes Page 21.2

Alternative splicing
Some exons may be removed from primary transcript with introns Same RNA transcript can yield 1+ kinds of mature mRNA Regulated by proteins that bind to pre-mRNA, interact with spliceosomes 90% of human sequences affected; 20500 genes produce 50000+ proteins

MicroRNA (miRNA)
Small RNA molecules that silence expression of specific mRNA Effect called RNA interference (RNAi) Animals, plants, also in some bacteria Associates with cellular proteins to become RNAinducing silencing complex (RISC) RISCs affect specific mRNAs based on complementarity Either inhibits translation or degrades mRNA

Glucocorticoid
Hormone released after meals 1. Enters cytosol, binds to receptors 2. Chaperones released, expose nuclear localization signal (NLS) 3. Receptors dimerize, enter nucleus through pore 4. Dimer binds to response elements next to genes 5. Transcription activated, eventually leads to protein

Lecture Notes Page 21.3

Cells
October 19, 2012 10:00

Cell theory
1. 2. 3. 4. Cells Cells Cells Cells are the fundamental unit of life. are both distinct entities and building blocks of more complex organisms. are created from pre-existing cells by division. contain heritable material, which is maintained over division.

All cells probably descend from an ancestral cell from over a few billion years ago.

This fossil prokaryote is 3.5 B years old!

3 major domains of life

Prokaryotic cells
Typical E. coli

Eukaryotic cell
Typical animal cell

Lecture Notes Page 22.1

 Most important feature is internal compartmentalization (not just membrane-enveloped nucleus, but also other organelles)

Often multicellular

Endosymbiosis theory How mitochondria came to be

Evidence for endosymbiosis: Size of mitochondria similar to typical bacterium, replicates by fission M have their own ribosomes to manufacture own proteins M have double membranes; consistent w/ engulfing M have their own genomes; genes code for enzymes needed to replicate & transcribe own genomes

Lecture Notes Page 22.2

Protein Traffic
October 24, 2012 10:00

Three fundamentally different ways

1. Gated transport: between cytosol & nucleus Two spaces topologically equivalent b/c "liquid" is continuous Nuclear pores = selective gates, actively transport macromolecules Smaller molecules freely diffuse 2. Transmembrane transport: membrane-bound protein translocators transport proteins from cytosol into topologically distinct space Transported protein must unfold to go through e.g. cytosol -> mitochondria 3. Vesicular transport: membrane-enclosed transport intermediates are vehicles that ferry proteins from compartment <-> compartment Small, spherical or larger, irregular Pinch off from source's membrane Fuse with destination compartment Compartments are topologically equivalent e.g. proteins from ER -> Golgi

Sorting signals
Choice of mechanism guided by sorting signals in protein Recognized by complementary sorting receptor proteins (e.g. part of translocator) Receptors generally recognize classes of proteins, not specific

First type: continuous signal sequence ~1560 residues long Specifies destination Once sorting is done, some are excised by signal peptidases Used in cytosol -> ER/mitochondria/chloroplasts/peroxisomes (i.e. transmembrane transport, where proteins have to unfold to pass through) Also used in nucleus -> cytosol, Golgi -> ER Second type: 3D atom arrangement in folded structure (signal patch) Often discontinuous in primary AA sequence Persist in finished protein, not removed Used in cytosol -> nucleus Also used to move new degradative enzymes -> lysosomes Complex, cannot be transferred from protein to protein for vesicular transport

Lecture Notes Page 23.1

experimentation

Organelle Duplication
Daughter cells inherit complete set of specialized membranes; cannot construct such membranes from scratch New ER cannot be made without existing ER; same for mitochondria, plastids, peroxisomes Information for organelles not exclusively in DNA Epigenetics (1+ protein already in organelle membrane required, passed from parent to progeny in organelle)

Lecture Notes Page 23.2

Endoplasmic Reticulum
October 24, 2012 10:00

Membrane is > of total membrane in animal cell Net-like, branching tubules, flattened sacs; all interconnect to form a single internal space (ER lumen) ER membrane compartmentalizes lumen, mediates selective transfer of molecules

Function
Lipid, protein biosynthesis Membrane = site of production For all transmembrane proteins, lipids for most organelles Most of lipids in mitochondrial/peroxisomal membranes Proteins to be secreted to extracellular destinations, or going to Golgi/lysosomes first go to lumen

Rough ER
ER captures proteins from cytosol while they are synthesized 1. Transmembrane proteins only partly translocated across membrane, become embedded Generally destined for another membrane 2. Water-soluble proteins are fully translocated into lumen Destined for lumen of an organelle or secretion In mammalian cells, import into ER is co-translational (concurrent) Mitochondria, chloroplasts, nuclei, peroxisomes import proteins post-translation One end of protein in ER while rest is made, so protein will not fold Chaperones not required to keep unfolded Ribosome translating such a protein is directly attached to ER membrane Region is called rough ER Ribosomes can be membrane-bound (rough ER) or free ribosomes Same structure, function Differ only in protein products Only mRNA that encodes protein with ER signal sequence bind to rough ER membranes All others in cytosol, translated by free ribosomes

Smooth ER
No bound ribosomes Contain ER exit sites where transport vesicles bud off -> Golgi Prominent in cells specializing in lipid metabolism Also in Ca2+ sequestering; ions stored in lumen by binding proteins Relevant proteins on specialized smooth ER, like Ca2+-ATPas in muscles

Signal-Recognition Particle (SRP)

ER signal sequence guided to ER membrane by SRP and SRP receptor in membrane

Lecture Notes Page 24.1

 SRP moves between membrane and cytosol, binds to signal sequence Structurally, six different polypeptides bound to single RNA Binding site is large hydrophobic pocket with methionines ER signal sequences vary but all have 8+ nonpolar AAs at center Directs ribosome to the membrane SRP-ribosome complex binds to SRP receptor, close to protein translocator Growing polypeptide chain transferred across membrane

Polypeptide goes through aqueous pore in translocator

Translocator is Sec61 complex of ~12 proteins together in a donut structure Hole aligns with tunnel in large ribosomal subunit, seals tightly Signal sequence probably triggers opening of pore Signal sequence recognized: a. First by SRP b. Then by binding site in translocator

Signal sequence sometimes removed after translocation

Removed if N-terminal

Lecture Notes Page 24.2

 Retained if internal within polypeptide chain

Single-pass transmembrane proteins

Structurally, signal sequence spans membrane as helix. Case 1: N-terminal SS + internal SS Have a single internal signal sequence within the bilayer Might stop translocation process after the N-terminal signal sequence initiates translocation N-terminal signal sequence is cleaved Case 2: internal SS, C-terminus in lumenal side Leaves N-terminus outside ER in cytosol C-terminus inside lumen Case 3: internal SS, N-terminus in lumenal side Leaves N-terminus inside ER in lumen C-terminus outside in cytosol

Multi-pass transmembrane proteins

Multiple signal sequences, probably starting with internal SS not N-terminus Distinction between start and stop based on order Since all membrane proteins inserted from cytosolic side, all copies of same polypeptide chain will always have same orientation Polypeptide passes back and forth repeatedly across bilayer

Translocated polypeptides fold, assemble in rough ER lumen

Some proteins are ER resident proteins that are kept there by an ER retention signal (4 AAs at C-terminus) Protein disulfide isomerase (PDI) catalyzes oxidation of free SH groups to form disulfide bonds BiP chaperone, recognizes incorrectly folded proteins and subunits not yet assembled into complexes Pulls proteins post-translationally into ER through translocator using ATP energy Binds exposed amino acid sequences normally sequestered in interior Keeps protein from aggregating, keeps in ER, out of secretory pathway

Bad proteins sent to cytosol for degradation

Retrotranslocation called dislocation, also occurs via same Sec61 complex Misfolded protein is deglycosylated, then fed to proteasomes to degrade the protein

Lecture Notes Page 24.3

Vesicular Traffic
October 26, 2012 10:00

Compartments within cell specialized based on combinations of membrane markers

Coats
Transport vesicles form from membranes Bud off as coated vesicles with a cage of proteins on surface Before fusing with target membrane, the coat is discarded Coat has two functions 1. Concentrates specific membrane proteins in a patch that leads to the vesicle membrane Involved in selecting package for transport 2. Assembly of proteins into curved lattices deforms the membrane patch, molds vesicle

Three main types, differing in proteins 1. Clathrin-coated From Golgi / from plasma membrane 2. COPI-coated From ER and Golgi cisternae 3. COPII-coated From ER and Golgi cisternae Formation of clathrin coat drives vesicle formation Major protein = clathrin 3 large, 3 small subunits -> three-legged structure triskelion Form hexagons, pentagons for pits Isolated triskelions spontaneously assemble into polyhedral cages Second protein = multisubunit adaptin complex Binds clathrin to membrane Traps transmembrane proteins including cargo receptors that interact with soluble proteins inside Different kinds of adaptin for different cargo receptors Assembly of adaptins and clathrin coat -> lateral interactions lead to bud formation

Lecture Notes Page 25.1

Endocytosis
Inward from cell surface -> lysosomes Intake of macromolecules, particles, sometimes cells Progressively enclosed by part of plasma membrane, which then invaginates, pinches off into endocytic vesicle Distinguished by size of vesicle 1. Phagocytosis (large, e.g. microorganisms, dead cells) via phagosomes, >250 nm diameter Specialized phagocytic cells 2. Pinocytosis (small, e.g. fluid and solutes), ~100 nm Most cells constantly do this Pinocytic vesicles Continually internalized from plasma membrane, then returned Endocytosis and exocytosis must be linked because SA & V remain relatively constant Endocytic cycle: Begins at clathrin-coated pits (short lifetime) Coated vesicles even more transient, within seconds, shed coat & fuse with endosomes Anything dissolved in encapsulated fluid is internalizedfluid-phase endocytosis Receptor-mediated endocytosis Macromolecules bind to complementary transmembrane receptor proteins, accumulate in coated pits, then enter as receptor-macromolecule complexes Selective concentrating mechanism Increases efficiency Minor extracellular elements can be taken in without large volume of fluid Best example: cholesterol When uptake blocked, builds up in blood => atherosclerotic plaques Most cholesterol is cholestryl ester -> cholesterol low-density lipoprotein (LDL) When cell needs LDL, makes more receptors for LDL on plasma membrane Receptors diffuse until close to pits Receptor binds to LDL, then both enclosed in coated vesicle LDL & receptor released when encountering acidic pH in endosomes Lysosomes hydrolyze to make free cholesterol Genetic defects: a. Lacking receptor b. Defective receptor (external binding site, or internal attachment to pit) 25+ different receptors Some preferentially associate with pits when bound to ligand

Lecture Notes Page 25.2

 Signal peptides guide transmembrane proteins to coated pits, bind to adaptins Endosomes Differ in protein composition Early endosomes near plasma membrane Late endosomes near Golgi and near nucleus Interior kept acidic pH 6 by H+-ATPase Pumps H+ into lumen Later endosomes more acidic Some endocytosed materials diverted from pathway back to plasma membrane Molecules not diverted -> lysosome for degradation Endocytosed receptors Some endocytosed ligands remain bound to receptors, follow fate of receptors Different receptors treated differently 1. Most recycled back to same plasma membrane domain 2. Some return to different plasma membrane domain = transcytosis 3. Some go to lysosomes for degradation LDL receptor follows first pathway Transferrin Soluble protein carrying iron in blood 1. Transferrin receptor binds with transferrin 2. Endocytosis 3. Low pH in endosome causes iron to be released 4. Transferrin & transferrin receptor recycled to plasma membrane 5. Transferrin -> exocytosed

Viruses
Enveloped viruses enter host by fuse with plasma membrane (e.g. HIV) or endosomal membrane (e.g. influenza)

Nonenveloped viruses form a pore in cell membrane (e.g. polio) or disrupt endosomal membrane (e.g. adenovirus)

Lecture Notes Page 25.3

Cytoskeleton
October 31, 2012 10:00

How do things move around quickly when they are large?

Microtubules
Microtubule overview
Polarity (+, ends) arbitrarily defined not by charge Minus ends at centrosome in centre Plus ends toward outside

Molecular motors
Carry cargo either in plus (kinesin) or minus (dynein) direction along MT Transport organelles Use ATP Mechanical cycle (bind to MT, power stroke = step, unbind) coupled with chemical cycle (ATP hydrolysis)

Kinesin takes steps about 8 nm apart 2 m/sec = more lengths per second than a gasoline race car Smaller force than gasoline engine, but more efficient!

Lecture Notes Page 26.1

Microtubule structure

Hollow in center Tubulin binds GTP, has GTPase activity Subunit = heterodimer of alpha, beta tubulin monomers Subunits together make protofilament 13 parallel protofilaments make microtubule Tubulin binds GTP; catalyzes GTP hydrolysis

Dynamic instability
Transitions between growing and shrinking Growing = adding GTP Shrinking = GDP form, unstable end Competition between adding GTP and hydrolyzing GTP->GDP

Lecture Notes Page 26.2

Lecture Notes Page 26.3