Desire For DNA Flashcards
Why do we need DNA
Need stable info storage
Problems with RNA as a storage molecule
- spontaneous deamination of cytosine into uracil cannot be recognised and repaired in RNA, making it unstable
- 2’OH makes unstable
What is the rate of cytosine deamination ?
- 1/16,250 cytosines daily
- 200 events per human cell per day
What are the two chemical differences between RNA and DNA?
- extra methyl on T in DNA
- no 2’OH deoxyribose
What does the extra methyl on T mean?
Deamination of C can be detected
Why is the reverse complement good?
Increased information stability - mispairing displays mutations for detection and correction
Describe the double helix
- 0.34nm per base
- right handed helix
- major groove = 2.2nm
- minor grove = 1.2nm
- 3.5nm per turn
- 10.4bp per turn
How is dNTP synthesised?
- RNR removes 2’OH from NDPs
- RNR makes dADP, dGDP, dCDP and dUDP
- dTDP made from dUDP
RNR
ribonucleotide reductase
NDPs
Nucleoside diphosphates
Evidence that DNA evolved from RNA
- Deoxyribose is made from ribose
- Thymine is made from uracil
DNA Polymerase catalyses
- addition of dNTP into 3’OH
- creates a new 3’OH, a phosphodiester bond, and releases pyrophosphate
Properties of DNA Polymerase
- single stranded DNA template
- elongation of RNA primer
- dNTP building blocks
- unidirectional synthesis (5’->3’)
- remains on ssDNA template (processive, β-clamp)
- some have proof reading activity
Proofreading in polymerase
polymerase reverses when it senses an incorporated mismatch
Without proofreading, there is
1 error per 10^5 copied nucleotides
With proofreading, there is
1 error per 10^7 copied nucleotides
With additional mismatch repair, there is
1 error per 10^9 copied nts
Describe mismatch repair
- catalysed by different DNA polymerases
- without proofreading it doesn’t progress
What are the repair polymerases in prokaryotes?
Pol I, II, IV, V
What are the repair polymerases in eukaryotes?
Pol-ν, Pol-μ, Pol-λ, Pol-κ, Pol-ι, Pol-θ, Pol-η, Pol-ζ, Pol-β
What are the replication polymerases in prokaryotes?
Pol-III
What are the replication polymerases in Eukaryotes?
Pol-α, Pol-δ, Pol-ε
Describe the enzymes involved in mismatch repair
- DNA glycosilase
- Endonuclease
- DNA Polymerase
- Ligase
What is the fork rate in transcription
1000bp/sec
How big is an Okazaki fragment?
1000bp
Lagging strand replication
- Primase binds helicase to create 11nt RNA primers
- DNA polymerase makes Okazaki fragments
- Rnase H degrades RNA primers
- Different DNA polymerases extend Okazaki to a Glenys
- DNA Ligase joins adjacent Okazaki fragments
Both lagging and leading polymerases are in the
Same dynamic protein complex
Replication in prokaryotes
- can be continuous
- single origin
- circular DNA
Describe the structure of prokaryotic DNA
θ
Describe replication in eukaryotes
- in S phase
- multiple origins
- linear chromosome
What does the Christmas tree structure of transcription under EM illustrate?
Multiple polymerases per gene
Describe RNA polymerase
- NTP building blocks
- unidirectional synthesis (5’->3’)
- ds DNA template
- only copied template strand
- start from initiation site, without primer
- regulated, complex initiation procedure
Where does the energy for DNA unwinding and RNA synthesis come from?
Pyrophosphate release
Transcription initiation in prokaryotes
- DNA motifs recognised by RNAP σ factor
- RNAP unwinds transcription bubble
Describe DNA transcription initiation motifs
- two of them
- upstream of transcription initiation site
- -35 and -10
What do we label the transcription initiation site?
+1
RNA Synthesis in prokaryotes
- σ factor dissociates
- RNAP elongates 5’->3’
RNAP
RNA Polymerase
Transcription bubble
17bp DNA
RNA Polymerase
- 450kDa tetramer
- 4 subunits (α2ββ’)
What are the three main RNA polymerases in eukaryotes?
- Pol-I
- Pol-II
- Pol-III
Which RNA does Pol-I polymerise?
rRNA
Which RNA does Pol-II polymerise?
mRNA (snRNA, miRNA)
Which RNA does Pol-III polymerise?
tRNA, 5s rRNA
What is the TATA box
A core promoter
How far are the enhancer sequences in DNA of the promoter
> 1kb upstream
TBP
- TATA binding protein
- one of the 9 subunits of TFIID
TFIID
Transcription Factor IID
Describe transcription initiation in eukaryotes
- TBP binds to TATA box
- TBP recruits TFIIA, TFIIB
- TFIIB recruits Pol-II and TFIIF
- TFIIE joins and recruits TFIIH
- Formation of transcription bubble, Pol-II phosphorylated
- Pol-II dissociâtes from TFIID, Pol-II transcribes RNA 5’-3’
What does eukaryotic DNA contain that allows transcription initiation?
- TATA box
- Upstream regulatory elements in promoter
- Enhancer sequences
TFIIH
helicase/kinase
How is phosphorylation of Pol-II achieved
signals from upstream regulatory elements
Describe the 5’ end of transcribed RNA in prokaryotes
- pppG/A: triphosphate purine
Describe the 5’ end of transcribed RNA in eukaryotes
- 5’Cap (7mGppp)
- (often) 2’O methylation on first/second nt
What is 5’Cap needed for?
- splicing
- translation
- increased RNA stability
What is the 5’ Cap?
N7-methyl-guanidine-5’-triphosphate
What are the types of transcription termination in prokaryotes
- Rho-independent termination
- Rho-dépendent termination
UTR
untranslated region
Where is the termination signal in prokaryotic transcription termination?
In the 3’UTR of the mRNA
Describe Rho-independent termination
- terminating GC-rich hairpin folds in 3’UTR
- then low affinity U-rich region
- NusA binds hairpin to terminate RNAP
Describe Rho-dependent termination
- Rho binds to rut
- Moves to 3’ to terminate RNAP
rut
Rho-utilisation site
Describe transcription termination in eukaryotes
- Cleavage by end ONU lease downstream of AAUAAA box
- Poly-A polymerase adds PolyA tail
Describe the PolyA tail
- 200-250nt A
- increases RNA stability
What is splicing?
- Removal of intron sequences from preRNA
- frequently in eukaryotes, occasionally in prokaryotes
preRNA
precursor RNA
What catalyses splicing?
Spliceozome (ribozyme)
What entities does splicing involve?
- 5 snRNAs with proteins
- self-splicing introns
snRNAs
small nuclear RNAs
In prokaryotes, transcription and translation can occur
Simultaneously
Why can transcription and translation not occur simultaneously in eukaryotes
Extra regulatory steps: 5’Cap, PolyA, splicing and export
Compare and contrast the templates between replication and transcription
Replication: ssDNA
Transcription: dsDNA
Compare and contrast the products between replication and transcription
Replication: dsDNA
Transcription: preRNA
Compare and contrast the monomers between replication and transcription
Replication: dNTPs
Transcription: NTPs
Compare and contrast the enzymes between replication and transcription in prokaryotes
Replication: DNA Pol-III
Transcription: RNAP
Compare and contrast the enzymes between replication and transcription in eukaryotes
Replication: Pol-α, Pol-δ, Pol-ε
Transcription: RNA Pol-II
Compare and contrast the initiation between replication and transcription
Replication: at origin, uses RNA primer
Transcription: at initiation site
Compare and contrast the structure between replication and transcription
Replication: replication fork
Transcription: transcription bubble
Compare and contrast the unique features between replication and transcription
Replication: proofreading
Transcription: regulation
Describe cytosine deamination
- spontaneous chemical mutation
- RNA decays into a more uracil rich sequence
RNA is relatively stable, but
Not as stable as DNA
Why does DNA not fold?
It cannot create 2’ H-bonds
Why is uracil substitution recognised in DNA?
- U is not a DNA base
- recognised by copy strand
Why are nitrogenous base pairs positive ?
They accept hydrogen
DNTP
deoxyribonucleic triphosphate
What is the RNA primer extended with?
ssDNA
What does a promoter do?
Regulates transcription
What does a kinase do?
Phosphorylate
