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Recall. Replication is semi-conservative and bidirectional. transcription. translation. Central Dogma. DNA. RNA. Protein. replication. Discussing DNA replication (Nucleus of eukaryote, cytoplasm of prokaryote). Lecture 10. DNA Replication. *Leading and lagging strand synthesis.

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central dogma

Recall

Replication is semi-conservative and bidirectional

transcription

translation

Central Dogma

DNA

RNA

Protein

replication

Discussing DNA replication

(Nucleus of eukaryote, cytoplasm of prokaryote)

lecture 10
Lecture 10

DNA Replication

*Leading and lagging strand synthesis

Biochemistry of replication

DNA mutation and repair

Transcription

dna polymerase adds nuclotides at 3 end of existing strand

3’ end

template

ECB 6-10

5’ end

DNA polymerase -adds nuclotides at 3’ end of existing strand

3’ OH

Incoming nucleotide

(triphosphate) adds at 3’OH

of growing chain (condensation

rx driven by cleavage of PiPi)

Synthesis occurs

in 5’ - 3’ direction

Specificity of which base adds depends on base pairing

with template strand ( strands are complementary)

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

ECB 6-11

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

(~100 NTs/sec in humans)

leading and lagging strands
Leading and lagging strands

Replication fork is asymmetrical

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

Other Proteins at Replication Fork

06.5-DNA_replication_fork.mov

ECB 6-17

Leading strand

Lagging strand

lecture 108
Lecture 10

DNA Replication

Leading and lagging strand synthesis

Biochemistry of replication

*DNA mutation and repair

Transcription

mutations accumulate with age and cause cancer
Mutations accumulate with age and cause cancer

Cancer = loss of control

of the cell cycle

Incidents of cancer per 100,000

women

ECB 6-20

Age (years)

mutations and repair

Cell mechanism to

reduce:

proofreading

Cell mechanism to reduce:

Many mechanisms

of repair

Mutations and Repair

During Replication:

Incorrect nucleotide

incorporated

Post Replication:

Many sources of

DNA damage

proofreading

3’ end

5’ end

Proofreading

DNA Polymerase

polymerase

3’ OH

-Catalyzes phosphodiesterbond formation

-Has exonuclease

activity

Incoming deoxy-ribonucleoside triphosphate

-Performs proofreading

5’ end

proofreading mechanism

3’

5’

G

5’

A

Proofreading mechanism

T

Exonuclease activity removes the incorrect nucleotide

Polymerase activity then adds correct nucleotide

After proofreading, mistakes about 1/107 nucleotides

See ECB 6-13

proofreading requires 5 to 3 dna synthesis

Replication

5’ Triphosphate

Replication

3’ OH

PP

Proofreading requires 5’ to 3’ DNA synthesis

Energy from

pyrophosphate

Energy from PP

At 5’ end

Error

Error

Incorrect

dNTP removed

Incorrect

dNTP removed

No PPP left

At 5’ end

3’ OH available

Correct dNTPbut cannot beadded

Energy frompyrophosphate

Corrected!

ECB 6-15

mutations and repair16
Mutations and Repair

During Replication:

Incorrect nucleotide

incorporated

Post Replication:

Many sources of

DNA damage

Causes of Errors:

Deamination

Depurination

Pyrimidine dimers

Post-replication repair also removes

99% of errors made in replication;

Final error rate 1/109 NT

Cell mechanism to

reduce:

proofreading

Cell mechanisms to reduce:

Many, involve removing

damaged DNA

post replication repair requires excision resynthesis ligation
Post-replication repair requires excision, resynthesis, ligation

How damaged strand is recognized is not understood

ECB 6-26

lecture 1018
Lecture 10

DNA Replication

Semi-conservative replication

Biochemistry of replication

End replication problem

DNA mutation and repair

*Transcription

central dogma19

transcription

translation

Central Dogma

DNA

RNA

Protein

Nucleus of eukaryote

Cytoplasm of prokaryote

replication

ECB 7-1

what is a gene

Transcription control regions

5’

3’

Coding region

3’

5’

What is a gene?

Double stranded DNA

Protein A

Protein B

Protein C

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

how much of genome is composed of genes
How much of genome is composed of genes?

Genome Projects:

Bacteria - about 500 genes, most of genome

Eukaryotes - 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 element

11% is Alu sequence, about 300 nucleotide pairs

rna structure

Single stranded

Variety of 3D structures

Ribonucleotides

AUCG

Organized as RNA-proteincomplexes

DNA Structure:

Double stranded

Double helix

Deoxyribonucleotides

ATCG

Organized as chromatin

RNA Structure:
dna codes for rna
DNA codes for RNA

Single-stranded product

(5’ to 3’)

Not H bonded to DNA

RNA Polymerase

Coded by DNA template strand (also called antisense strand)

base pairing

DNA

template

A:U two

H bonds

New

NTP

Base pairing

Rate ~ 30 NT/sec

rna polymerases
RNA Polymerases

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.

lecture 1028
Lecture 10

DNA Replication

Semi-conservative replication

Biochemistry of replication

End replication problem

DNA mutation and repair

Transcription-general

*Prokaryotes

Eukaryotes

prokaryotic gene organization
Prokaryotic Gene Organization

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

One mRNA codes for

Several proteins

ECB 8-6

transcription initiation in bacteria overview

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

Transcription of one strand

Transcription Initiation in Bacteria - overview

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 initiation in bacteria cont d

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

transcription start site

+1 = transcription start site

Transcription Initiation in Bacteria (cont’d)

ECB 7-9

transcription termination in bacteria

ECB 7-9

Terminator (stop sequence)

Transcription termination in bacteria

RNA

Hairpin loop causes polymerase to fall off DNA

genome of mycoplasma genitalium
Genome of Mycoplasma genitalium

One of smallest genomes of any cell: codes for

470 proteins

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

gene regulation in prokaryotes

Operator sequence associated

with promoter

Gene regulation in prokaryotes

ECB 8-6

Regulatory protein (transcription factor)

binds operator

repressible operon
Repressible operon

Default state is on, can turned off

ECB 8-7

inducible operon lac operon
Inducible operon; lac operon

Default state is off, turned on