Discussing DNA replication (Nucleus of eukaryote, cytoplasm of prokaryote) Central Dogma DNA RNA Protein transcription translation replication Replication is semi-conservative and bidirectio Recall
Dec 20, 2015
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 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 6-20
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
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::
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
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
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