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: • 1920s–1940s: 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

Watson & Crick • Each strand of DNA has all info needed for copying Lecture Notes Page 16. 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.Replication September 28." .1 .

2 .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.

Note that these diagrams only show leading strands and not lagging strands. To be continued… Lecture Notes Page 16.3 .

Replication (continued) October 3. synthesizes short RNA segment that serves as primer. new DNA) slips through the cut • Cut is re-ligated In linear chromosomes (eukaryotes) • Larger chromosomes. Single-strand DNA-binding proteins (SSBPs) attach to separated strands to prevent closing 3. coli. about 80 million bp • Total human genome = 3.3 billion bp • DNA polymerases much slower.1 . Helicase uses ATP to separate strands 2. Unwinding creates tension down the helix. Lecture Notes Page 17. can reach 1000 bp/s Type II topoisomerases cut both strands in a duplex simultaneously • Second duplex (i. DNA polymerase requires primer—a few nucleotides bonded to template strand with a free 3' –OH group. Primase (RNA polymerase). so topoisomerase cuts one strand then rejoins strands downstream to relieve this tension 4. in humans.e. 2012 10:00 In closed circular chromosomes (prokaryotes) • • • • Single ori 1–5 million bp in chromosome DNA polymerases very fast. about 50 bp/s • Hundreds of ori in humans to increase speed of replication Leading strand synthesis • Leading strand = toward replication fork 1. in E.

• In humans. 5 different DNA polymerases. coli. chromosomal DNA replication is DNA polymerase Lagging strand synthesis • • • • Synthesized discontinuously in direction away from replication fork.• Remember that new bases are always added to 3' 5. responsible enzyme is DNA polymerase III. Synthesis of leading strand begins. 14 different DNA polymerases (some for error correction). 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. moving toward replication fork • Energy from energy of dNTPs • In E.2 .

Process repeats for multiple Okazaki fragments 4. DNA polymerase I removes primer. 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. then reaches primer and stops 3.3 .2. 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 polymerase III synthesizes first fragment.

adds bases to 3' end of lagging strand • 90% of human cancers also produce it Lecture Notes Page 17.Telomeres • Region at end of linear chromosomes = telomere • Leading strand synthesis is normal. 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. 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 50–200 bp with each replication • Usually cell dies after 20–30 generations Solution • Telomeres are non-encoding genes.4 .

5 .Lecture Notes Page 17.

1 . 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.Central Dogma October 5.

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. 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.1 .Transcription November 3.

Transcription process in prokaryotes 1.2 . -35 box is TTGACA • Sigma binds to -10 and -35 boxes • Different sigma proteins allow recognition of different promoters 2. 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. Initation phase continued: sigma opens double helix and threads template strand through active site ("open complex") Lecture Notes Page 19.

tRNA • RNA modified after transcription (pre-mRNA -> mRNA) RNA modification 1: splicing • Happens inside nucleus • Introns are non-coding.• Sigma dissociates from core enzyme once initiation is done 3. but each step has more proteins • RNA polymerase II: transcribes mRNA (or pre-mRNA) • Requires 5 factors instead of just sigma • RNA polymerase I. 7-methylguanosine • Needed for exit of mRNA from nucleus.3 . must be removed by splicing • Small nuclear ribonucleoproteins (snRNPs) form spliceosome. separates transcript from polymerase Transcription process in eukaryotes • Similar to prokaryotes. contain both amino and nucleic acids • Exons are coding regions. 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. III: transcribe non-structural genes for rRNA. interrupt genes. part of final mRNA product RNA modification 2: capping • 5' cap. Lecture Notes Page 19.

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. binding to ribosome for translation RNA modification 3: tailing • 3' tail. which is after the promoter. lac repressor binds to operator. 100–200 adenine nucleotides (AAAAAAAAAAAA…) • Also needed for export from nucleus • Increases stability. 7-methylguanosine • Needed for exit of mRNA from nucleus. can block polymerase • Positive control: inducing or increasing gene transcription • Activators Lecture Notes Page 19. or block it • In lac.RNA modification 2: capping • 5' cap.4 . lifespan • To remove all mRNA from cell.

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. 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. 2012 10:00 • mRNA is converted to a protein.1 .Translation October 10.

interacts directly & specifically with codon. transfer RNA (tRNA) • Aminoacyl tRNA synthetases charge tRNA by catalyzing addition of AAs to tRNAs • Must be highly accurate.2 . high fidelity • Use ATP to link amino acid Lecture Notes Page 20.Codon -> amino acid translation • Francis Crick: adapter molecule holds amino acids in place.

different aminoacyl-tRNAs enter but the one with the right anticodon stays Lecture Notes Page 20.3 . and come together during initiation General translation process 1.• tRNA covalently linked to corresponding AA is aminoacyl tRNA • Anticodon on tRNA makes contact with codon (recognition) Wobble hypothesis • 61 codons. anticodon can bind successfully to codon whose third position requires nonstandard base pairing (e. 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. Large subunit: peptide bonds form • They can separate. 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. 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.g. Elongation phase: initiator tRNA (f-Met) in P site • Charged tRNAs enter A site. 40 tRNAs in most cells • In some cases.

• Peptide bonds form between amino acids on the tRNAs.4 . A is empty and then this cycle continues 3. added to C-terminus of previous amino acid • Elongation factors move mRNA down 3 nucleotides at a time = translocation • tRNAs move A->P->E. Termination phase: when A site encounters stop codon • Release factor protein enters site (not tRNA). if E site already had a tRNA. it is ejected. no amino acid carried but shape resembles tRNA • Catalyze hydrolysis of tRNA in P site with polypeptide • Ribosome subunits separate Lecture Notes Page 20.

Gene Expression in Eukaryotes October 15. 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. Fibres form even more complex protein scaffold 4. several levels of organization 1. (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. 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. Other enzymes catalyze acetylation and methylation of histones ○ Acetylation (negatively-charged groups attached to positively-charged lysines) reduces positive charge. 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 • Chromatin has a regular structure. 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. Chromatin-remodeling complexes reshape chromatin ○ Use ATP 2. 2012 10:00 Various points of control affect gene expression (and modulate the level of gene expression). Nucleosomes together create 30-nm fibre 3.1 .

2 . 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.• Also elements far from promoter.

3 . plants. expose nuclear localization signal (NLS) 3. binds to receptors 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. 20500 genes produce 50000+ proteins MicroRNA (miRNA) • Small RNA molecules that silence expression of specific mRNA • Effect called RNA interference (RNAi) • Animals. Enters cytosol. Receptors dimerize. Dimer binds to response elements next to genes 5. enter nucleus through pore 4. 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. Chaperones released. eventually leads to protein Lecture Notes Page 21. Transcription activated. interact with spliceosomes • 90% of human sequences affected.

coli Eukaryotic cell • Typical animal cell Lecture Notes Page 22.Cells October 19. 4. This fossil prokaryote is 3. are both distinct entities and building blocks of more complex organisms. are created from pre-existing cells by division. Cells Cells Cells Cells are the fundamental unit of life. which is maintained over division. 2. All cells probably descend from an ancestral cell from over a few billion years ago. 3.5 B years old! 3 major domains of life Prokaryotic cells • Typical E. contain heritable material.1 . 2012 10:00 Cell theory 1.

genes code for enzymes needed to replicate & transcribe own genomes Lecture Notes Page 22. replicates by fission • M have their own ribosomes to manufacture own proteins • M have double membranes.2 .• 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. consistent w/ engulfing • M have their own genomes.

g. Transmembrane transport: membrane-bound protein translocators transport proteins from cytosol into topologically distinct space • Transported protein must unfold to go through • e.Protein Traffic October 24. part of translocator) • Receptors generally recognize classes of proteins.e.g. not removed • Used in cytosol -> nucleus • Also used to move new degradative enzymes -> lysosomes • Complex. Gated transport: between cytosol & nucleus • Two spaces topologically equivalent b/c "liquid" is continuous • Nuclear pores = selective gates. cytosol -> mitochondria 3. where proteins have to unfold to pass through) • Also used in nucleus -> cytosol. spherical or larger. irregular • Pinch off from source's membrane • Fuse with destination compartment • Compartments are topologically equivalent • e. actively transport macromolecules • Smaller molecules freely diffuse 2. transmembrane transport. Golgi -> ER Second type: 3D atom arrangement in folded structure (signal patch) • Often discontinuous in primary AA sequence • Persist in finished protein. not specific First type: continuous signal sequence • ~15–60 residues long • Specifies destination • Once sorting is done. some are excised by signal peptidases • Used in cytosol -> ER/mitochondria/chloroplasts/peroxisomes (i. 2012 10:00 Three fundamentally different ways 1. Vesicular transport: membrane-enclosed transport intermediates are vehicles that ferry proteins from compartment <-> compartment • Small. proteins from ER -> Golgi Sorting signals • Choice of mechanism guided by sorting signals in protein • Recognized by complementary sorting receptor proteins (e. cannot be transferred from protein to protein for vesicular transport Lecture Notes Page 23.g.1 .

2 . passed from parent to progeny in organelle) Lecture Notes Page 23. same for mitochondria. peroxisomes • Information for organelles not exclusively in DNA • Epigenetics (1+ protein already in organelle membrane required. plastids. cannot construct such membranes from scratch • New ER cannot be made without existing ER.experimentation Organelle Duplication • Daughter cells inherit complete set of specialized membranes.

nuclei. 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. import into ER is co-translational (concurrent) • Mitochondria. lipids for most organelles ○ Most of lipids in mitochondrial/peroxisomal membranes • Proteins to be secreted to extracellular destinations. Transmembrane proteins only partly translocated across membrane. protein biosynthesis • Membrane = site of production ○ For all transmembrane proteins. 2012 10:00 • Membrane is > of total membrane in animal cell • Net-like. or going to Golgi/lysosomes first go to lumen Rough ER • ER captures proteins from cytosol while they are synthesized 1. mediates selective transfer of molecules Function • Lipid.1 . chloroplasts. Water-soluble proteins are fully translocated into lumen ○ Destined for lumen of an organelle or secretion • In mammalian cells. 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.Endoplasmic Reticulum October 24. function • Differ only in protein products • Only mRNA that encodes protein with ER signal sequence bind to rough ER membranes • All others in cytosol. ions stored in lumen by binding proteins • Relevant proteins on specialized smooth ER. flattened sacs. branching tubules. 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. all interconnect to form a single internal space (ER lumen) • ER membrane compartmentalizes lumen. become embedded ○ Generally destined for another membrane 2.

seals tightly • Signal sequence probably triggers opening of pore • Signal sequence recognized: a.• SRP moves between membrane and cytosol. Then by binding site in translocator Signal sequence sometimes removed after translocation • Removed if N-terminal Lecture Notes Page 24. 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.2 . binds to signal sequence • Structurally. 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. First by SRP b.

3 . keeps in ER. 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. then fed to proteasomes to degrade the protein Lecture Notes Page 24. N-terminus in lumenal side • Leaves N-terminus inside ER in lumen • C-terminus outside in cytosol Multi-pass transmembrane proteins • Multiple signal sequences. 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. probably starting with internal SS not N-terminus • Distinction between start and stop based on order • Since all membrane proteins inserted from cytosolic side. signal sequence spans membrane as helix. all copies of same polypeptide chain will always have same orientation • Polypeptide passes back and forth repeatedly across bilayer Translocated polypeptides fold. also occurs via same Sec61 complex • Misfolded protein is deglycosylated.• Retained if internal within polypeptide chain Single-pass transmembrane proteins • Structurally. 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. out of secretory pathway Bad proteins sent to cytosol for degradation • Retrotranslocation called dislocation.

Clathrin-coated ○ From Golgi / from plasma membrane 2. the coat is discarded • Coat has two functions 1.1 . 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. Assembly of proteins into curved lattices deforms the membrane patch.Vesicular Traffic October 26. 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. 3 small subunits -> three-legged structure triskelion • Form hexagons. differing in proteins 1. Concentrates specific membrane proteins in a patch that leads to the vesicle membrane ○ Involved in selecting package for transport 2. COPII-coated ○ From ER and Golgi cisternae Formation of clathrin coat drives vesicle formation • Major protein = clathrin • 3 large. COPI-coated ○ From ER and Golgi cisternae 3. molds vesicle • Three main types.

particles. 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. fluid and solutes). 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. shed coat & fuse with endosomes • Anything dissolved in encapsulated fluid is internalized—fluid-phase endocytosis Receptor-mediated endocytosis Macromolecules bind to complementary transmembrane receptor proteins. dead cells) via phagosomes. ~100 nm ○ Most cells constantly do this Pinocytic vesicles • Continually internalized from plasma membrane. then both enclosed in coated vesicle ○ LDL & receptor released when encountering acidic pH in endosomes • Lysosomes hydrolyze to make free cholesterol • Genetic defects: a.Endocytosis • Inward from cell surface -> lysosomes • Intake of macromolecules. e. builds up in blood => atherosclerotic plaques • Most cholesterol is cholestryl ester -> cholesterol ∈ low-density lipoprotein (LDL) • When cell needs LDL. pinches off into endocytic vesicle • Distinguished by size of vesicle 1. 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. accumulate in coated pits. Phagocytosis (large. makes more receptors for LDL on plasma membrane ○ Receptors diffuse until close to pits ○ Receptor binds to LDL. sometimes cells • Progressively enclosed by part of plasma membrane.g. e. within seconds. >250 nm diameter ○ Specialized phagocytic cells 2.2 . microorganisms. Pinocytosis (small. which then invaginates. Lacking receptor b.g.

Transferrin -> exocytosed • • • • Viruses • Enveloped viruses enter host by fuse with plasma membrane (e. Transferrin & transferrin receptor recycled to plasma membrane 5. influenza) • Nonenveloped viruses form a pore in cell membrane (e. Some go to lysosomes for degradation LDL receptor follows first pathway Transferrin • Soluble protein carrying iron in blood 1. Some return to different plasma membrane domain = transcytosis 3. HIV) or endosomal membrane (e.g.g. 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.g. Endocytosis 3. adenovirus) Lecture Notes Page 25.• Signal peptides guide transmembrane proteins to coated pits. Most recycled back to same plasma membrane domain 2. Low pH in endosome causes iron to be released 4.3 . polio) or disrupt endosomal membrane (e.g. Transferrin receptor binds with transferrin 2.

but more efficient! Lecture Notes Page 26. 2012 10:00 • How do things move around quickly when they are large? Microtubules Microtubule overview • Polarity (+.Cytoskeleton October 31. – 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.1 . unbind) coupled with chemical cycle (ATP hydrolysis) • Kinesin takes steps about 8 nm apart • 2 μm/sec = more lengths per second than a gasoline race car • Smaller force than gasoline engine.

Microtubule structure • Hollow in center • Tubulin binds GTP. unstable end • Competition between adding GTP and hydrolyzing GTP->GDP Lecture Notes Page 26. has GTPase activity • Subunit = heterodimer of alpha. beta tubulin monomers • Subunits together make protofilament • 13 parallel protofilaments make microtubule • Tubulin binds GTP.2 . catalyzes GTP hydrolysis Dynamic instability • Transitions between growing and shrinking • Growing = adding GTP • Shrinking = GDP form.

Lecture Notes Page 26.3 .

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