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7/31/2019 Lecture Notes for 2nd Midterm
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Nucleic AcidsNucleic acids = polymers (RNA, DNA)
Phosphate group
Five-carbon sugar (ribose/deoxyribose), carbons numbered from 1' where base is
Cytosine (C)
Uracil (U)
Thymine (T)
Pyrimidines (single ring):
Guanine (G)
Adenine (A)
Purines (double ring):
Single/double ring of carbon & nitrogen atoms, nitrogenous base
Monomer is nucleotide
RNA is made up ofribonucleotide monomers
DNA deoxyribonucleotide monomers
Structure of chain
Nucleotides added to 3' end when polymerizing
Sugar-phosphate backbone is directional: 5' end (3' carbon unlinked), 3' end (3' C unlinked)
Written in the 5' -> 3' direction
Genetic materialMust contain information for entire organism
Must be accurately copied
Should account for known variation within, without species
1920s1940s: expected protein part of chromosomes to be genetic material
History:
DNASeptember 26, 2012 10:04
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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
Demonstrated transformation of bacteria from DNA
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
Measured where radioactivity was; experiment 1: radioactive phosphorus was in pellet, experiment 2:
radioactive sulphur was in supernatant
Hershey, Chase (1952): tested whether protein or DNA in bacteriophage was responsible for genetic material
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
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DNA StructureStrands antiparallel in double helix
Hydrophilic sugar-phosphate backbone faces exterior
Nitrogenous base pairs face interior
Major groove
Minor groove
Two different sized grooves in helix (i.e. not symmetric)
A-T 2 H-bonds
C-G 3 H-bonds
Binding sites on C=O groups and N groups
Purines pyrimidines
Stabilized byhydrophobic interactions in interior
as well as H-bonding between complementary base pairs
Base pairs are exposed in grooves
RNAAlso sugar-phosphate backbone, four nitrogenous bases
Uracil (U) instead of thymine (T)1.
Presence of additional OH means RNA is less reactive, less stable
Ribose instead of deoxyribose2.
Differs from DNA in 2 ways:
Can be a catalytic molecule; ribozymes are enzyme-like RNAs
Full genetic material
Unlike DNA, can function like a protein
Theory: early life originated with RNA
Secondary structure of RNAComplementary base pairing
Typically forms H-bonds between bases on the same strand
Often observe hairpin single-stranded RNA
Nucleic acids (continued)September 28, 2012 10:00
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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
ReplicationSeptember 28, 2012 10:30
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What is the mechanism?
Demonstrated by Kornberg (1956), who found DNA polymerase I
Part of large replication complex
DNA polymerase catalyzes replication
Unidirectional polymerization: each base on the template strand gains a dNTP (deoxyribonucleoside
triphosphate) (the form of a free base)
Replication complex binds to ori
Replication occurs in both directions from ori, forming two replication forks
All chromosomes have origin of replication (ori)
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In closed circular chromosomes (prokaryotes)Single ori
15 million bp in chromosome
DNA polymerases very fast; in E. coli, can reach 1000 bp/s
Second duplex (i.e. new DNA) slips through the cut
Cut is re-ligated
Type II topoisomerases cut both strands in a duplexsimultaneously
In linear chromosomes (eukaryotes)
Total human genome = 3.3 billion bp
Larger chromosomes, about 80 million bp
DNA polymerases much slower; in humans, about 50 bp/s
Hundreds ofori in humans to increase speed of replication
Leading strand synthesisLeading strand = toward replication fork
Helicase uses ATP to separate strands1.
Single-strand DNA-binding proteins (SSBPs) attach to separated strands to prevent closing2.
Unwinding creates tension down the helix, so topoisomerase cuts one strand then rejoins strands downstream
to relieve this tension
3.
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.
4.
Replication (continued)October 3, 2012 10:00
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DNA polymerase III synthesizes first fragment, then reaches primer and stops2.
Process repeats for multiple Okazaki fragments3.
Exonuclease (removes primers)
DNA polymerase
Two enzymatic activities:
Uses 3' end of next Okazaki fragment as primer for new dNTPs
DNA polymerase I removes primer, replaces with deoxyribonucleotides4.
Leaves gap between former RNA primer and the Okazaki fragment
DNA ligase closes the gap in the sugar-phosphate backbone5.
Most of these enzymes around the replication fork are probably in one large multi-enzyme machine: replisome
(replication complex)
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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 moveA->P->E, ifEsite already had a tRNA, it is ejected;A is empty and then this cycle continues
Release factor protein enters site (not tRNA), no amino acid carried but shape resembles tRNA
Catalyze hydrolysis of tRNA in Psite with polypeptide
Ribosome subunits separate
Termination phase: whenA site encounters stop codon3.
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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 compactingDNA wrapped around proteins to create a protein-DNA
complex, chromatin
negatively-charged DNA wrapped twice around
eight positive-charged histone proteins
histone H1 maintains structure of each
nucleosome
linker DNA between nucleosomes
Nucleosomes are beadlike structures1.
Nucleosomes together create 30-nm fibre2.
Fibres form even more complex protein scaffold3.
Everything condenses to chromosome4.
Chromatin has a regular structure, several levels of
organization
Opening up chromatin
Condensed chromatin -> open chromatin
DNase degrades open chromatin to fragments but
leaves condensed chromatin intact
Chromatin must be relaxed/decondensed for
transcription
Use ATP
Chromatin-remodeling complexes reshape
chromatin
1.
histone acetyl transferases (HATs)
histone deacetylases (HDACs)
Acetylation (negatively-charged groups
attached to positively-charged lysines)
reduces positive charge; associated with
activation
Methylation ~ activation or inactivation
Other enzymes catalyze acetylation and
methylation of histones
2.
Two types of proteins
An example ofepigenetic inheritance; not
due to differences in gene sequences
Daughter cells inherit patterns
Histone code hypothesis: chemical modifications of
histones contain information influencing gene expression
Transcription control
Also have gene-unique promoter-proximal element
Promoters etc similar to prokaryotes
Gene Expression in EukaryotesOctober 15, 2012 10:00
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Also elements farfrom promoter; DNA looping etc allow
them to have effects even though they are far
Enhancers (positive control)
upstream/downstream/within introns
Silencers (negative control) shut down transcription
Regulatory sequences that affect gene transcription
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Alternative splicing
Same RNA transcript can yield 1+ kinds of
mature mRNA
Some exons may be removed from primary transcript
with introns
Regulated by proteins that bind to pre-mRNA, interact
with spliceosomes
90% of human sequences affected; 20500 genes
produce 50000+ proteins
MicroRNA (miRNA)
Effect called RNA interference (RNAi)
Small RNA molecules that silence expression of
specific mRNA
Animals, plants, also in some bacteria
RISCs affect specific mRNAs based on
complementarity
Associates with cellular proteins to become RNA-
inducing silencing complex (RISC)
Either inhibits translation or degrades mRNA
GlucocorticoidHormone released after meals
Enters cytosol, binds to receptors1.
Chaperones released, expose nuclear localization
signal (NLS)
2.
Receptors dimerize, enter nucleus through pore3.
Dimer binds to response elements next to genes4.
Transcription activated, eventually leads to protein5.
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Cell theoryCells are the fundamental unit of life.1.
Cells are both distinct entities and building blocks of more complex organisms.2.
Cells are created from pre-existing cells by division.3.
Cells contain heritable material, which is maintained over division.4.
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 cellsTypical E. coli
Eukaryotic cellTypical animal cell
CellsOctober 19, 2012 10:00
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experimentation
Organelle Duplication
New ER cannot be made without existing ER; same for
mitochondria, plastids, peroxisomes
Daughter cells inherit complete set of specialized membranes; cannot
construct such membranes from scratch
Epigenetics (1+ protein already in organelle membrane
required, passed from parent to progeny in organelle)
Information for organelles not exclusively in DNA
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Compartments within cell specialized based on combinations of membrane markers
Coats
Bud off as coated vesicles with a cage of proteins on surface
Before fusing with target membrane, the coat is discarded
Transport vesicles form from membranes
Involved in selecting package for transport
Concentrates specific membrane proteins in a patch that leads to the vesicle membrane1.
Assembly of proteins into curved lattices deforms the membrane patch, molds vesicle2.
Coat has two functions
From Golgi / from plasma membrane
Clathrin-coated1.
From ER and Golgi cisternae
COPI-coated2.
From ER and Golgi cisternaeCOPII-coated3.
Three main types, differing in proteins
Formation of clathrin coat drives vesicle formation
3 large, 3 small subunits -> three-legged structure triskelion
Form hexagons, pentagons for pits
Isolated triskelions spontaneously assemble into polyhedral cages
Major protein = clathrin
Binds clathrin to membrane
Traps transmembrane proteins including cargo receptors that interact with soluble proteins inside
Different kinds of adaptin for different cargo receptors
Second protein = multisubunit adaptin complex
Assembly of adaptins and clathrin coat -> lateral interactions lead to bud formation
Vesicular TrafficOctober 26, 2012 10:00
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Signal peptides guide transmembrane proteins to coated pits, bind to adaptins
Endosomes
Early endosomes near plasma membrane
Late endosomes near Golgi and near nucleus
Differ in protein composition
Pumps H+ into lumen
Later endosomes more acidic
Interior kept acidic pH 6 byH+-ATPase
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
Most recycled back to same plasma membrane domain1.
Some return to different plasma membrane domain = transcytosis2.
Some go to lysosomes for degradation3.
Different receptors treated differently
LDL receptor follows first pathway
Soluble protein carrying iron in blood
Transferrin receptor binds with transferrin1. Endocytosis2.
Low pH in endosome causes iron to be released3.
Transferrin & transferrin receptor recycled to plasma membrane4.
Transferrin -> exocytosed5.
Transferrin
VirusesEnveloped 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) ordisrupt endosomal membrane (e.g.
adenovirus)
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How do things move around quickly when they are large?
MicrotubulesMicrotubule overview
Minus ends at centrosome in centrePlus ends toward outside
Polarity (+, ends) arbitrarily defined not by charge
Molecular motors
Transport organelles
Mechanical cycle (bind to MT, power stroke = step, unbind) coupled with
chemical cycle (ATP hydrolysis)
Use ATP
Carry cargo either in plus (kinesin) or minus (dynein) direction along MT
2 m/sec = more lengths per second than a gasoline race car
Smaller force than gasoline engine, but more efficient!
Kinesin takes steps about 8 nm apart
CytoskeletonOctober 31, 2012 10:00
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