Biochemistry IV

DNA

informational molecule

sequence of basses specifies genetic information

• within genes, nucleotides specify the amino acid sequences of proteins

• outside genes, regulatory sequences direct DNA replication, mRNA synthesis

polymer of deoxyribonucleotides

deoxyribonucleotides

PICTURE 5

nucleotide linkage

phosphodiester bonds

3’ links to 5’ in the 5’→3’ direction

PICTURE 6

hydrogen bond lengths

2.6-3 Angstroms

base-pairing interactions

hydrogen bonding

complementary between a purine and pyrimidine (A/T, C/G)

PICTURE 8

DNA structure

two strands held together by base-pairing interactions

strands are antiparallel

sugar-phosphate backbone on the outside

bases are stacked on inside

complementary allows information to be copied

PICTURE 9

double helix

right handed helix

has major and minor grooves

structure stabilized by hydrogen bonding and stacking interactions

PICTURE 10

B form DNA

most stable

found in solution

10.5 bp per turn

right handed

PICTURE 14

A form DNA

DNA-RNA, RNA-RNA helix

found insolution

11 bp per turn

right handed

PICTURE 14

Z form DNA

left handed helix

PICTURE 14

DNA denaturation

high temperature or pH

partial denaturation then full separation

denaturation → increased UV absorbance due to disruption of base stacking interactions

• hyper chromic effect

tm = temperature at which 50% of the DNA is random coil (not helical)

PICTURE 16

RNA structure

single strand helix

contains secondary structure elements

• single strands

• bulge

• internal loop

• hairpin

PICTURE 18

RNase P structure

G can also pair to U

PICTURE 19

hydrogen bonding opportunities DNA

bond acceptors and donors are available in

• the major groove (AT, TA, GC, CG can be discriminated

• the minor groove (AT/TA vs. GC/CG can be discriminated

PICTURE 22

amino acid side chains and DNA

form hydrogen bonds with bases in double helical DNA

• Asparagine

• Glutamine

• Glutamate

• Lysine

• Arginine

sequence-specific DNA recognition is key to carrying out the steps in information transfer

PICTURE 23

DNA binding proteins

generally bind DNA in the major groove

• alpha-helix fits nicely into the wide major groove

certain DNA binding motifs are common

• helix-turn-helix (HTH)

• zinc finger

• homeodomain

• leucine zipper

• basic helix-loop-helix (BHLH)

helix-turn-helix

recognition helix is positioned in the major groove

domains in the lac repressor protein

PICTURE 25/26

zinc finger

coordinated Zn2+ ion

PICTURE 27

homeodomain

widely conserved DNA binding motif of ~60 amino acids

alpha-helix positioned in the major groove

PICTURE 28

leucine zipper

Leu residues stabilize dimerization

Lys and Arg residues: DNA binding, in the major groove

PICTURE 29

helix-loop-helix

basic residues mediate DNA binding

amphipathic helices mediate dimerization

PICTURE 30

DNA supercoiling

relieves strain

when strands separated, helix is no longer relaxed

intrinsic property of DNA tertiary structure

occurs in all cellular DNAs and is highly regulated by each cell

PICTURE 34

polymerases in supercoiling

separates the strands and introduce strain

PICTURE 36

underwound DNA

most cellular DNA is underfund: supercoiling stabilizes underwinding

PICTURE 38

Lk

topological linking number

separation of two strands of a double-stranded circular DNA (Lk = 1)

specifies the number of helical turns in a closed circular DNA

PICTURE 39

Lk and DNA

Lk = #bps / bps per turn

superhelical density = σ

σ = deltaLk/Lk0

PICTURE 40

positive and negative supercoils

two forms of a circular DNA that differ only in a topological property such as linking number are referred to as topoisomers

PICTURE 41

topoisomerases

act to change the linking number of DNA

two types

topoisomerase 1

change Lk in increments of 1

cleave one strand of duplex DNA

can remove negative supercoils

PICTURE 43

topoisomerase 1 mechanism

PICTURE 44/45

topoisomerase 2

changes Lk in increments of 2

cleaves both strands of DNA

can relax positive and negative supercoils

can introduce negative supercoils (prokaryotes only)

hydrolyzes ATP

decatenates DNA circles

examples: DNA gyrase, topoisomerase II

PICTURE 48

topoisomerase 2 mechanism

PICTURE 47

plectonemic and solenoidal supercoiling

does not produce sufficient compaction to package DNA in the cell

PICTURE 49

DNA packaged

into nucleosomes

around histone core

causes supercoiling

long non-coding RNAs and associated proteins also organized DNA in chromosomes

PICTURE 51/57

histones

package DNA

found in chromatin of all eukaryotic cells

nucleosome core particle

2 each of histones H2A, H2B, H3, H4

146 bp of DNA wrapped around histone core

PICTURE 53

histone tails

amino terminal tails of histone proteins protrude from the core particles

these tails are extensively post-translationally modified and also participate in DNA packaging

PICTURE 55

histone modifications

HFD: histone fold domain

PICTURE 56

higher order DNA organization

higher magnification imaging shows DNA attached in loops to scaffold protein

extraction of histone proteins reveals a protein-based chromosomal scaffold

PICTURE 58

chromosome organization

active and inactive compartments

binding by CTC-binding factor (a protein) organizes loops into topologically associated domains (TAD)

inactive regions are called heterochromatin

constrained in chromosome territories in nucleus

PICTURE 59/61

cohesins and condensins

organize DNA in eukaryotic cell cycle

PICTURE 62

DNA polymerase require:

template strand to copy

primer strand with 3’ OH

dNTP substrates

Catalyzes:

• nucleophilic attack by 3’ OH

• phosphodiester bond formation

• 5’→3’ synthesis

DNA polymerase activity

PICTURE 6

replication base pair geometry

important for binding in active site

DNA polymerases insert one incorrect nucleotide for every 10^4 to 10^5 correct nucleotides

PICTURE 7

DNA proofreading

3’→5’ exonuclease activity for this

• DNA polymerases insert one incorrect nucleotide for every 10^4 to 10^5 correct nucleotides

• proofreading improves the inherant accuracy of the polymerization reaction by 100- to 1000- fold

• in combination, one ent error for every 10^6-10^8 bases added

PICTURE 8

dna replication initiation

requires specific sequences at replication origin

DUE = DNA unwinding element (contains high amount AT)

PICTURE 11/12

DNA elongation: priming

primase places RNA primers to create a small loop in DNA strand at origin

PICTURE 14

DNA elongation: polymerization

DNA polymerase III synthesizes 5’→3’ from the two RNA primers

PICTURE 15

elongation: DNA replication

semidiscontinuous

leading strand synthesis is continuous and in direction of fork movement

lagging strand synthesis is discontinuous and opposite to fork movement

PICTURE 16

replisome

combination of all replication machinery

PICTURE 17

DNA elongation steps

leading strand: Primase synthesizes an RNA primer at the origin

Lagging strand: primase synthesizes an RNA primer for each Okazaki fragment

2. dNTPs are added by DNA polymerase III

3. as replication fork moves

   1. DnaB helicase unwinds DNA

   2. SSB stabilizes the single strands

   3. DNA gyrase relieves the strain caused by unwinding

4. RNA primers are removed by DNA polymerase I and the nicks are clsoed by DNA ligase

DNA pol III enzyme

PICTURE 19

beta sliding clamp

on DNA pol III and slides along DNA to keep moving and attached to the strand

PICTURE 20

elongation: lagging strand synthesis

has a clamp loading complex

PICTURE 21

DNA synthesis overview picture

PICTURE 22

DNA pol III clamp loader

PICTURE 23

removal of RNA primers

PICTURE 24

DNA pol I

removes RNA primers

5’→3’ exonuclease activity, removes RNA

5’→3’ polymerase activity, fills in with DNA

PICTURE 25

DNA ligase

seals the nick

Phosphodiester bond formation

1. adenylylation of enzyme

2. activation of 5’ phosphate

3. nucleophilic attack by 3’ OH

PICTURE 26

replication termination

replication forks stop at the terminus region

PICTURE 28

topoisomerase IV

decatenates the two chromosomes

PICTURE 29

eukaryotic chromosomes

long and linear

multiple origins of replication are necessary to replicate large chromosomes

chromosomes must be replicated only once per cell cycle

PICTURE 33

pre-replicative complex

assembles at eukaryotic origin

PICTURE 32

telomerase

adds telomeres to chromosome ends

• telomerase synthesizes DNA from an RNA template (reverse transcriptase)

• template is an RNA molecule that is part of the enzyme

• telomerase is a ribonucleoprotein (RNP)

PICTURE 35

retrovirus

utilizes RNA-dependent DNA synthesis to make viral DNA which is integrated with integrase and then proteins formed (HIV)

PICTURE 37

human LINE-1 retrotransposon

contributes to age associated diseases

PICTURE 38

DNA mutations

permanent change in DNA sequence

can be:

• silent → no effect on gene function

• deleterious → impairs gene function

• advantageous → enhances gene function

can lead to:

• genetic diversity

• cancer in somatic cells

• birth defects in germ cells

PICTURE 41

deamination

spontaneously, ~100/day, good reason for having T instead of U in DNA

deaminating agents induce these conversions at high levels

PICTURE 42

deaminating agents

nitrous acid precursors

• NaNO2

• NaNO3

• nitrosamine

metabolized to nitrous acid (HNO2), a strong deaminating agent

depurination

removes purines

can occur:

• spontaneously

• through the action of aklylating agents

• N7 alkylation increases depurination

PICTURE 44

UV irradiation

another source of DNA damage

defects in repair of this lesion lead to Xeroderma pigmentosum

generates a block in replication
PICTURE 45

alkylating agents

dimethylnitrosamine

dimethylsulfate

nitrogen mustard

S-adenosylmethionine

PICTURE 46

methylation of G

by dimethylsulfate produces O6-methylguanine

PICTURE 47

alkylation

can change base-pairing properties

this can lead to permanent mutations

can also be repaired

PICTURE 48/49

mismatch repair

allows correction of replication errors

methylation distinguishes between template and newly synthesized strands

PICTURE 51

mismatch repair mechanism

exonueclase activity degrades DNA from methyl past mismatch

DNA pol III replaces DNA (copies methylated strand)

PICTURE 52/53

base excision repair

PICTURE 54

nucleotide-excision repair

used for removal of large bulky lesions (pyrimidine dimers)

excinuclease:

• excision endonuclease

• makes 2 cuts

• excises the damaged DNA

PICTURE 55

direct repair

does not remove base or nucleotide

repairs the defect directly but its expensive

Cost = one protein inactivated per repair

PICTURE 56

direct repair of alkylated bases by AlkB in e coli

oxidative demethylation by an alpha-ketoglutarate-iron dependent dioxygenase

PICTURE 57

rna classes

mRNA - messenger RNA

rRNA - ribosomal RNA

tRNA - transfer RNA

snRNA - small nuclear RNA

snoRNA - small nucleolar RNA

siRNA - small interfering RNA

miRNA - micro RNA

piRNA - kiwi-interacting RNA (germline specific)

transcription vs replication

all RNA molecules (except some viruses) are derived from information permanently stored in DNA

during replication, entire genome usually copied

during transcription, only particular genes or groups of genes are transcribed at one time (some portions never transcribed)

only one DNA strand serves a template for transcription

RNA polymerase

don’t require a primer

cannot perform 3’→5’ exonuclease activity so no proofreading

PICTURE 5

rna synthesis initiation

initiated by promoters

RNA polymerases recognize and bind to specific DNA sequences called promoters

PICTURE 11

sigma subunits

enable coordinated gene expression on RNA polymerase holoenzyme

recognizes the two spacers before the RNA start

PICTURE 12/13

e coli transcription

1. RNA polymerase core and sigma70 subunit bind to DNA promoter

2. transcription bubble forms

3. transcription is initiated

4. promoter clearance is followed by elongation

5. elongation continues. sigma70 dissociates and is replaced by NusA

6. transcription is terminated. NusA dissociates, and the RNA polymerase is recycled

PICTURE 14

transcription termination rho-independent

RNA hairpin forms at palindromic sequence and disrupts interactions between RNA and DNA template within polymerase releasing the mRNA

PICTURE 15

transcription termination rho-dependent

rho helicase separates the mRNA from the DNA template

rut: rho utilization

PICTURE 16

operons

only in prokaryotes

transcription produces polycistronic mRNAs

PICTURE 17

rna polymerases in eukaryotes

Pol I → rRNA

Pol II → mRNA, snRNA

Pol III → tRNA, 5S rRNA

consensus RNA polymerase II promoter

in general, eukaryotic promoters have little intrinsic affinity for RNA polymerase

PICTURE 19

RNAP II trascription

requires transcription factors

sequential assembly at promoter:

• TFs, RNAP II, more TFs

closed complex: >30 proteins

open complex: TFIIH unwinds DNA

initiation:

• phosphorylation of CTD

• phosphodiester bond formation

• promoter clearance

PICTURE 20

RNA pol II CTD phosphorylation

carboxyl-terminal domain

PICTURE 21

eukaryotic transcription and RNA processing

PICTURE 25

5’ capping

enhances stability, only in eukaryotes, roles in processing and translation

PICTURE 27

capping enzymes

tethered to Pol II CTD

in transcription and RNA processing

four mechanisms of RNA splicing

group I introns - self-splicing; catalytic RNA

group II introns - self-splicing; catalytic RNA

spliceosome - catalytic snRNA + proteins

protein-catalyzed - splicing endonuclease + ligase

ribozymes

group I and II introns

RNA molecules with catalytic activities

some:

• group I introns

• RNase P

• hammerhead ribozyme

common activities:

• phosphodiester bond cleavage

• transesterification

PICTURE 31/32

group II intron splicing mechanism

PICTURE 33

small nuclear RNPs

assemble into the spliceosome

for example, the U1 snRNP recognizes the 5’ splice site by base pairing

RNP = ribonucleoprotein

PICTURE 34