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Michaelmas Term 1 > Desire For DNA > Flashcards

Desire For DNA Flashcards

1
Q

Why do we need DNA

A

Need stable info storage

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2
Q

Problems with RNA as a storage molecule

A
  • spontaneous deamination of cytosine into uracil cannot be recognised and repaired in RNA, making it unstable
  • 2’OH makes unstable
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3
Q

What is the rate of cytosine deamination ?

A
  • 1/16,250 cytosines daily
  • 200 events per human cell per day
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4
Q

What are the two chemical differences between RNA and DNA?

A
  • extra methyl on T in DNA
  • no 2’OH deoxyribose
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5
Q

What does the extra methyl on T mean?

A

Deamination of C can be detected

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6
Q

Why is the reverse complement good?

A

Increased information stability - mispairing displays mutations for detection and correction

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7
Q

Describe the double helix

A
  • 0.34nm per base
  • right handed helix
  • major groove = 2.2nm
  • minor grove = 1.2nm
  • 3.5nm per turn
  • 10.4bp per turn
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8
Q

How is dNTP synthesised?

A
  • RNR removes 2’OH from NDPs
  • RNR makes dADP, dGDP, dCDP and dUDP
  • dTDP made from dUDP
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9
Q

RNR

A

ribonucleotide reductase

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10
Q

NDPs

A

Nucleoside diphosphates

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11
Q

Evidence that DNA evolved from RNA

A
  1. Deoxyribose is made from ribose
  2. Thymine is made from uracil
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12
Q

DNA Polymerase catalyses

A
  • addition of dNTP into 3’OH
  • creates a new 3’OH, a phosphodiester bond, and releases pyrophosphate
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13
Q

Properties of DNA Polymerase

A
  • 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
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14
Q

Proofreading in polymerase

A

polymerase reverses when it senses an incorporated mismatch

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15
Q

Without proofreading, there is

A

1 error per 10^5 copied nucleotides

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16
Q

With proofreading, there is

A

1 error per 10^7 copied nucleotides

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17
Q

With additional mismatch repair, there is

A

1 error per 10^9 copied nts

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18
Q

Describe mismatch repair

A
  • catalysed by different DNA polymerases
  • without proofreading it doesn’t progress
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19
Q

What are the repair polymerases in prokaryotes?

A

Pol I, II, IV, V

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20
Q

What are the repair polymerases in eukaryotes?

A

Pol-ν, Pol-μ, Pol-λ, Pol-κ, Pol-ι, Pol-θ, Pol-η, Pol-ζ, Pol-β

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21
Q

What are the replication polymerases in prokaryotes?

A

Pol-III

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22
Q

What are the replication polymerases in Eukaryotes?

A

Pol-α, Pol-δ, Pol-ε

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23
Q

Describe the enzymes involved in mismatch repair

A
  1. DNA glycosilase
  2. Endonuclease
  3. DNA Polymerase
  4. Ligase
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24
Q

What is the fork rate in transcription

A

1000bp/sec

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25
Q

How big is an Okazaki fragment?

A

1000bp

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26
Q

Lagging strand replication

A
  1. Primase binds helicase to create 11nt RNA primers
  2. DNA polymerase makes Okazaki fragments
  3. Rnase H degrades RNA primers
  4. Different DNA polymerases extend Okazaki to a Glenys
  5. DNA Ligase joins adjacent Okazaki fragments
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27
Q

Both lagging and leading polymerases are in the

A

Same dynamic protein complex

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28
Q

Replication in prokaryotes

A
  • can be continuous
  • single origin
  • circular DNA
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29
Q

Describe the structure of prokaryotic DNA

A

θ

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30
Q

Describe replication in eukaryotes

A
  • in S phase
  • multiple origins
  • linear chromosome
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31
Q

What does the Christmas tree structure of transcription under EM illustrate?

A

Multiple polymerases per gene

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32
Q

Describe RNA polymerase

A
  • NTP building blocks
  • unidirectional synthesis (5’->3’)
  • ds DNA template
  • only copied template strand
  • start from initiation site, without primer
  • regulated, complex initiation procedure
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33
Q

Where does the energy for DNA unwinding and RNA synthesis come from?

A

Pyrophosphate release

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34
Q

Transcription initiation in prokaryotes

A
  1. DNA motifs recognised by RNAP σ factor
  2. RNAP unwinds transcription bubble
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35
Q

Describe DNA transcription initiation motifs

A
  • two of them
  • upstream of transcription initiation site
  • -35 and -10
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36
Q

What do we label the transcription initiation site?

A

+1

37
Q

RNA Synthesis in prokaryotes

A
  1. σ factor dissociates
  2. RNAP elongates 5’->3’
38
Q

RNAP

A

RNA Polymerase

39
Q

Transcription bubble

A

17bp DNA

40
Q

RNA Polymerase

A
  • 450kDa tetramer
  • 4 subunits (α2ββ’)
41
Q

What are the three main RNA polymerases in eukaryotes?

A
  • Pol-I
  • Pol-II
  • Pol-III
42
Q

Which RNA does Pol-I polymerise?

A

rRNA

43
Q

Which RNA does Pol-II polymerise?

A

mRNA (snRNA, miRNA)

44
Q

Which RNA does Pol-III polymerise?

A

tRNA, 5s rRNA

45
Q

What is the TATA box

A

A core promoter

46
Q

How far are the enhancer sequences in DNA of the promoter

A

> 1kb upstream

47
Q

TBP

A
  • TATA binding protein
  • one of the 9 subunits of TFIID
48
Q

TFIID

A

Transcription Factor IID

49
Q

Describe transcription initiation in eukaryotes

A
  1. TBP binds to TATA box
  2. TBP recruits TFIIA, TFIIB
  3. TFIIB recruits Pol-II and TFIIF
  4. TFIIE joins and recruits TFIIH
  5. Formation of transcription bubble, Pol-II phosphorylated
  6. Pol-II dissociâtes from TFIID, Pol-II transcribes RNA 5’-3’
50
Q

What does eukaryotic DNA contain that allows transcription initiation?

A
  1. TATA box
  2. Upstream regulatory elements in promoter
  3. Enhancer sequences
51
Q

TFIIH

A

helicase/kinase

52
Q

How is phosphorylation of Pol-II achieved

A

signals from upstream regulatory elements

53
Q

Describe the 5’ end of transcribed RNA in prokaryotes

A
  • pppG/A: triphosphate purine
54
Q

Describe the 5’ end of transcribed RNA in eukaryotes

A
  • 5’Cap (7mGppp)
  • (often) 2’O methylation on first/second nt
55
Q

What is 5’Cap needed for?

A
  • splicing
  • translation
  • increased RNA stability
56
Q

What is the 5’ Cap?

A

N7-methyl-guanidine-5’-triphosphate

57
Q

What are the types of transcription termination in prokaryotes

A
  1. Rho-independent termination
  2. Rho-dépendent termination
58
Q

UTR

A

untranslated region

59
Q

Where is the termination signal in prokaryotic transcription termination?

A

In the 3’UTR of the mRNA

60
Q

Describe Rho-independent termination

A
  • terminating GC-rich hairpin folds in 3’UTR
  • then low affinity U-rich region
  • NusA binds hairpin to terminate RNAP
61
Q

Describe Rho-dependent termination

A
  1. Rho binds to rut
  2. Moves to 3’ to terminate RNAP
62
Q

rut

A

Rho-utilisation site

63
Q

Describe transcription termination in eukaryotes

A
  1. Cleavage by end ONU lease downstream of AAUAAA box
  2. Poly-A polymerase adds PolyA tail
64
Q

Describe the PolyA tail

A
  • 200-250nt A
  • increases RNA stability
65
Q

What is splicing?

A
  • Removal of intron sequences from preRNA
  • frequently in eukaryotes, occasionally in prokaryotes
66
Q

preRNA

A

precursor RNA

67
Q

What catalyses splicing?

A

Spliceozome (ribozyme)

68
Q

What entities does splicing involve?

A
  • 5 snRNAs with proteins
  • self-splicing introns
69
Q

snRNAs

A

small nuclear RNAs

70
Q

In prokaryotes, transcription and translation can occur

A

Simultaneously

71
Q

Why can transcription and translation not occur simultaneously in eukaryotes

A

Extra regulatory steps: 5’Cap, PolyA, splicing and export

72
Q

Compare and contrast the templates between replication and transcription

A

Replication: ssDNA
Transcription: dsDNA

73
Q

Compare and contrast the products between replication and transcription

A

Replication: dsDNA
Transcription: preRNA

74
Q

Compare and contrast the monomers between replication and transcription

A

Replication: dNTPs
Transcription: NTPs

75
Q

Compare and contrast the enzymes between replication and transcription in prokaryotes

A

Replication: DNA Pol-III
Transcription: RNAP

76
Q

Compare and contrast the enzymes between replication and transcription in eukaryotes

A

Replication: Pol-α, Pol-δ, Pol-ε
Transcription: RNA Pol-II

77
Q

Compare and contrast the initiation between replication and transcription

A

Replication: at origin, uses RNA primer
Transcription: at initiation site

78
Q

Compare and contrast the structure between replication and transcription

A

Replication: replication fork
Transcription: transcription bubble

79
Q

Compare and contrast the unique features between replication and transcription

A

Replication: proofreading
Transcription: regulation

80
Q

Describe cytosine deamination

A
  • spontaneous chemical mutation
  • RNA decays into a more uracil rich sequence
81
Q

RNA is relatively stable, but

A

Not as stable as DNA

82
Q

Why does DNA not fold?

A

It cannot create 2’ H-bonds

83
Q

Why is uracil substitution recognised in DNA?

A
  • U is not a DNA base
  • recognised by copy strand
84
Q

Why are nitrogenous base pairs positive ?

A

They accept hydrogen

85
Q

DNTP

A

deoxyribonucleic triphosphate

86
Q

What is the RNA primer extended with?

A

ssDNA

87
Q

What does a promoter do?

A

Regulates transcription

88
Q

What does a kinase do?

A

Phosphorylate

Michaelmas Term 1 (42 decks)

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