lecture notes for 2nd midterm

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

Lecture Notes Page 14.1


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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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lecture notes for 2nd midterm

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