Discussing DNA replication(Nucleus of eukaryote, cytoplasm of prokaryote)

Central Dogma

DNA RNA Proteintranscription translation

replication

Replication is semi-conservative and bidirectionalRecall

Lecture 10

DNA Replication*Leading and lagging strand synthesis

Biochemistry of replication

DNA mutation and repair

Transcription

ECB 6-103’ end

5’ end

Incoming nucleotideIncoming nucleotide(triphosphate) adds at 3’OH (triphosphate) adds at 3’OH

of growing chain (condensationof growing chain (condensationrx driven by cleavage of PiPi)rx driven by cleavage of PiPi)

template

3’ OHDNA

polymerase -adds

nuclotides at 3’ end of

existing strandSynthesis occursin 5’ - 3’ direction

Specificity of which base adds depends on base pairingSpecificity of which base adds depends on base pairingwith template strand ( strands are complementary)with template strand ( strands are complementary)

Fork moves at rate of ~1000 nucleotides/sec in prokaryotes

(~100 NTs/sec in humans)

Problem: Strands are antiparallel; DNA made only 5’ to 3’

ECB 6-11

Leading and lagging strands

Replication fork is asymmetrical

Lagging strand synthesis occurs from RNA primer

ECB 6-16

Leading strandLeading strand

Lagging strandLagging strand

Other Proteins at Replication Fork

Helicase unwinds DNA duplex; breaks H bonds; requires ATP

Single stranded binding proteins coat strand and prevent renaturation

Sliding clamp keeps DNA polymerase bound

ECB 6-17 06.5-DNA_replication_fork.mov

Lecture 10

DNA ReplicationLeading and lagging strand synthesis

Biochemistry of replication

*DNA mutation and repair

Transcription

ECB 6-19

Mutation: a permanent change in

DNA sequence

ECB 6-20

Inci

den

ts o

f ca

nce

r per

100

,00

0

wom

en

Age (years)

Mutations accumulate with age and cause cancer

Cancer = loss of controlof the cell cycle

During Replication:Incorrect nucleotide

incorporated

Post Replication:Many sources of

DNA damage

Cell mechanism to reduce:

proofreading

Cell mechanism to reduce:

Many mechanismsof repair

Mutations and Repair

Mistakes during replication cause mutations

ECB 6-21

5’ end

5’ end

3’ end

3’ OH

Incoming deoxy-ribonucleoside triphosphate

polymerase

DNA Polymerase

-Catalyzes phosphodiesterbond formation

-Performs proofreading

Proofreading

-Has exonucleaseactivity

5’3’

5’

TG

Exonuclease activity removes the incorrect nucleotide

A

Proofreading mechanism

Polymerase activity then adds correct nucleotide

After proofreading, mistakes about 1/107 nucleotidesSee ECB 6-13

Replication3’ OH

Energy frompyrophosphate

Error Error

Incorrect dNTP removed

Incorrect dNTP removed

3’ OH available

Energy frompyrophosphate

Corrected!

Replication5’ Triphosphate

Energy from PPAt 5’ end

No PPP leftAt 5’ end

Correct dNTPbut cannot be

added

ECB 6-15

Proofreading requires 5’ to 3’ DNA synthesis

PP

During Replication:Incorrect nucleotide

incorporated

Post Replication:Many sources of

DNA damage

Cell mechanism to reduce:

proofreading

Cell mechanisms to reduce:

Many, involve removingdamaged DNA

Causes of Errors:DeaminationDepurination

Pyrimidine dimers

Mutations and Repair

Post-replication repair also removes99% of errors made in replication;Final error rate 1/109 NT

Post-replication repair requires excision, resynthesis, ligation

How damaged strand is recognized is not understood

ECB 6-26

Lecture 10

DNA ReplicationSemi-conservative replication

Biochemistry of replication

End replication problem

DNA mutation and repair

*Transcription

Central Dogma

Nucleus of eukaryoteCytoplasm of prokaryote

DNA RNA Proteintranscription translation

replication

ECB 7-1

5’

5’

3’

3’

Double stranded DNA

Protein AProtein A Protein BProtein B Protein CProtein C

“each gene contains the information required to make a protein”

Transcription control regions

Coding region

What is a gene?What is a gene?

How much of genome is composed of genes?

Genome Projects:Bacteria - about 500 genes, most of genomeEukaryotes - about 20,000-40,000 genes, represents much less of genome

Humans - about 30,000 genes, only a few percent of the total genome!!!Rest is repetitive DNA sequences - junk DNA Much of repetitive DNA is transposable elements that have mutated and can no longer move

15% of human genome is the L1 element11% is Alu sequence, about 300 nucleotide

pairs

Single strandedVariety of 3D structures

Ribonucleotides

AUCGOrganized as RNA-protein

complexes

DNA Structure:Double stranded

Double helixDeoxyribonucleotides

ATCGOrganized as chromatin

RNA StructureRNA Structure::

RNA Structure

ECB 7-3

Coded by DNA template strand (also called antisense strand)

RNA Polymerase

Single-stranded product(5’ to 3’)

Not H bonded to DNA

DNA codes for RNA

DNAtemplate

New NTP

Base pairing

A:U twoH bonds

Rate ~ 30 NT/sec

4 major RNA classes

Prokaryotes: One RNA Polymerase, composed of four subunits, plus additional factors that can confer promoter specificity

Eukaryotes: Three RNA Polymerases (RNA Pol I, II, III), each composed of >10 different proteins, transcribe different types of genes.

RNA Pol. I: synthesizes ribosomal RNA (rRNA)

RNA Pol II: synthesizes mRNA (protein coding) and some small RNAs.

RNA Pol III: synthesizes a variety of small RNAs, including tRNAs.

RNA Polymerases

Lecture 10

DNA ReplicationSemi-conservative replication

Biochemistry of replication

End replication problem

DNA mutation and repair

Transcription-general

*Prokaryotes Eukaryotes

Most genes in Operons: genes organized together, with one shared transcription start site

Prokaryotic Gene Organization

One mRNA codes forSeveral proteins

ECB 8-6

Promoter: “nucleotide sequence in DNA to which RNA polymerase binds and begins transcription.” ECB definition

RNA Polymerase contacts DNA, slides along strand, “looking” for promoter sequences.

At the promoter, the RNA Polymerase binds tightly, opening up a small single stranded region.

Transcription of one strand

Transcription Initiation in Bacteria - overview

Sigma subunit - binds RNA Pol., recognizes DNA sequencesin the promoter approximately 35 and 10 bases ‘upstream’ oftranscription start site

+1 = transcription start site

Transcription Initiation in Bacteria (cont’d)

ECB 7-9

Initiation and termination of prokaryotic transcription

ECB 7-9

Terminator (stop sequence)

RNA

Transcription termination in bacteria

Hairpin loop causes polymerase to fall off DNA

ECB 7-9

How does the cell know where to transcribe?… and when to transcribe?

Proteins bind to specific DNA sequences,Some activate transcription, some repress

Termed transcription factors

Genome of Mycoplasma genitalium

One of smallest genomes of any cell: codes for470 proteins

Regulatory Regulatory protein protein (transcription (transcription factor)factor)binds operatorbinds operator

Gene regulation in prokaryotes

ECB 8-6 Operator sequence associatedwith promoter

Repressible operonDefault state is on, can turned off

ECB 8-7

Inducible operon; lac operon

Default state is off, turned on