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MBB 331 > Lecture 12 > Flashcards

Lecture 12 Flashcards

1
Q

Why do we want to know the mechanism of transcription?

A

Since transcription is the first step of transferring genetic information to phenotypes

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2
Q

How is RNA different from DNA?

A
  • 2’-OH on nucleotide sugars
  • usually single-stranded but can have secondary structure
  • contains uracil instead of T as a base
  • RNA has short half-life
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3
Q

List the types of coding RNA. List two examples of non-coding RNA.

A 
Coding:
precursor messenger RNA (pre-mRNA)
messenger RNA (mRNA)
Non-coding:
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
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4
Q

How is the template different between replication and transcription?

A

Replication: both strands, entire strand
Transcription: template strand, selective

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5
Q

How is the primer different between replication and transcription?

A

Replication: DNA/RNA
Transcription: no primer

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6
Q

How is Polymerase different between replication and transcription?

A

Replication: DNA pol (one holoenzyme with proofreading)
Transcription: RNA pol (one in bacteria, three in eukaryote) without proofreading

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7
Q

How are nucleotides different between replication and transcription?

A

Replication: use dNTP
Transcription: use rNTP

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8
Q

How is synthesis different between replication and transcription?

A

Replication: semi-discontinuous
Transcription: processive and continuous

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9
Q

How are the products different between replication and transcription?

A

Replication: Replicon, daughter strands, DNA
Transcription: Primary transcript, RNA => mRNA, rRNA, tRNA

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10
Q

What ion is needed for both replication and transcriptioN?

A

Mg2+

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11
Q

What is the difference in synthesis directionality between replication and transcription?

A

There is none. Both are 5’ to 3’ synthesis

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12
Q

What is a transcription unit?

A

DNA sequence extending from promoter to open reading frame to terminator that is transcribed into a single RNA

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13
Q

What is another name for the +1 site?

A

Transcription start site (TSS)

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14
Q

What is an open reading frame?

A

ORF is the biologically relevant reading frame, ie. it is the DNA sequence from start codon to stop codon, that will be translated into a protein

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15
Q

What are the general steps to prokaryotic transcription?

A
  1. Recognition and binding
  2. Closed to open promoter melting
  3. Initiation (abortive)
  4. Elongation (promoter clearance)
  5. Termination and recycling
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16
Q

True or False: RNA pol has 5’ to 3’ exonuclease activity

A

False. RNAP only has 5’ to 3’ polymerase activity

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17
Q

What are the components of the prokaryotic RNAP core and their roles?

A

alpha2 - non-specific promoter recognition and assembly
beta + beta prime - catalytic subunits
omega - ?
sigma - promoter specificity

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18
Q

In the catalytic centre of the RNAP molecule, what amino acid coordinates the placement of the Mg2+ ions and how many of them are there?

A

Aspartic acid residues, 3

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19
Q

Why does it not matter that transcription has a high error rate?

A
  • Many copies of RNAs will be produced by one gene, the mismatch will only affect one molecule
  • RNA will be degraded soon, and reproduced when needed, while DNA mutation is permanent
  • Some surveillance mechanisms such as nonsense-mediated decay degrades mRNA with nonsense mutation
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20
Q

Where is the promoter region in E.coli?

A

-70 to + 30

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21
Q

What subunit of the RNAP core allows causes the RNAP molecule to bind tightly to a promoter?

A

Sigma factor

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22
Q

Where is the 10 box and what is its consensus sequence?

What is the box referred to as in prokaryotes? in eukaryotes?

A

10 bp upstream of transcription start site.
Consensus: TATAAT
Prokaryotes: pribnow box
Eukaryotes: TATA box

23
Q

Where is the 35 box and what is its consensus sequence?

A

35 bp upstream of transcription start site.

Consensus: TTGACA

24
Q

What are UP elements

A

Elements of the DNA that are bound by one alpha subunit and found in active promoters

25
Q

How many conserved regions are there in the sigma factor? What are their roles?

A

There are 4 conserved regions:
N-terminal region 1 - inhibit sigma 70 DNA binding in absence of core
Region 2 and 4 - contains -10 box and -35 box respectively. Contact point for two alpha-helix motifs

26
Q

List the factors affecting the efficiency of promoter binding by RNAP

A
  • The sequences in -35 box, -10 box and UP element
  • The spacing between -35 box, -10 box, and UP element
  • The distance between UP element and TSS
27
Q

What are “down” mutations? What is an example?

A

Mutations that reduce transcription.

Example: SNPs that cause divergence from consensus

28
Q

What are “up” mutations? What is an example?

A

Mutations that increase transcription.

Example: SNPS that cause a better match to the consensus

29
Q

What is DNase Footprinting? What is the protocol?

A

Method that investigates DNA/protein binding.
Protocol:
- Have 1 strand of DNA be end-labelled at 5’
- Incubate with protein of interest
- Partial digestion with endonuclease
- Digested DNA is run on polyacrylamide gel and visualized on x-ray film

30
Q

How do you interpret the gel from DNase footprinting?

A

Compare lanes with control lane. Missing bands denote DNA regions that are protected from nuclease digestion, so theses regions must be covered by the protein

31
Q

Alpha subunits of the RNAP core bind from -35 to -60 which includes the UP element (-40 to -60). What experiment can you do that would come to this conclusion?

A

DNAse footprinting on a strong promoter

32
Q

What are CTDs? What is their role?

A

CTDs (C-terminal domains) are domains of the alpha subunits of RNAP that bind specifically to the UP element and promotes very tight binding between polymerase and promoter

33
Q

What experimental evidence is there for the transition from the closed complex of DNA to open complex after RNAP binding?

A
  1. Elevated temperature stabilizes melted DNA and also stabilizes RNAP-promoter interaction
  2. E.coli RNAP binding to one of the phage T7 promoters causes hyperchromic shift indicative of 10bp strand separation
  3. Methylation-S1 nuclease experiments
34
Q

What is DMS footprinting? What is the protocol?

A

DMS (dimethyl sulfate) footprinting is another method to investigate DNA/Protein interactions.
Protocol:
- Have one of the DNA strands be labelled at the 5’ end
- Incubate with protein
- Methylate DNA with DMS
- Dislodge the protein and treat DNA with piperdine which removes methylated purines in DNA
- DNA fragments are then run through gel and autoradiographed

35
Q

What does a DMS footprinting assay show you that DNase footprinting does not?

A

DMS footprinting can detect distortion of DNA structure caused by protein binding and so has higher resolution than DNase footprinting

36
Q

What is DMS-S1 footprinting? What is the protocol?

A

DMS-S1 footprinting is another method to investigate DNA-protein interactions.
Protocol:
- end label promoter DNA
- incubate with RNAP (exposes adenines by promoter melting)
- incubate with DMS (methylate adenines)
- incubate with S1 nuclease sensitive (cuts methylated adenines)
- resolve fragments through gel at open regions
- sequence the fragments

37
Q

What does DMS-S1 footprinting assay show you that other assays do not?

A

Allows you to locate the region of the early promoter that has been melted by RNAP

38
Q

What are the two most common mechanisms of promoter melting?

A
  1. Sigma70 mediated melting

2. Sigma54 mediated melting

39
Q

Explain Sigma70 mediated promoter melting.

A

In complexes with sigma70-class factor:

  • the closed complex can spontaneously convert to a transcription-competent open complex, in a process of isomerization
  • Isomerization is reversible
40
Q

Explain Sigma54 mediated promoter melting.

A

Sigma54-containing holoenzymes require specialized activator proteins of AAA+ (ATPases associated with various cellular activities) family to catalyze conversion to the open complex with simultaneous ATP hydrolysis. Upstream activator sequences are brought into contact with promoter-bound sigma54-RNA polymerase through DNA looping, a step that is often facilitated by auxiliary DNA-binding and bending proteins sugh as integration host factor (IHF).
This process is irreversible and initiation must occur once it starts.

41
Q

List some characteristics of abortive initiation.

A
  • more likely to occur
  • results in aborted transcript (8-10 nts)
  • RNAP is close to transcription start site
  • restart RNA synthesis from the same position
42
Q

List some characteristics of promoter clearance:

A
  • happens occasionally
  • extended RNA (> 10 nts)
  • RNAP moves away from TSS
  • elongation phase of transcription
43
Q

Why does abortive initiation occur?

A

RNAP exit channel is blocked either by the RNAP itself or sigma subunit

44
Q

By what two mechanisms is transcription accuracy ensured?

A
  1. kinetic proofreading

2. nucleolytic proofreading

45
Q

Explain what occurs in kinetic proofreading.

A

Elongation stalls if the incorrect nucleotide is incorporated which allows pyrophophorolysis, a fast reaction

46
Q

Explain what occurs in nucleolytic proofreading.

A

Polymerase backtracks on DNA template melting several nucleotides and an intrinsic nuclease removes the melted nucleotides.

47
Q

What allows transcribed RNA to fold properly and be translated synchronously?

A

RNAP backtracking mechanism.
RNAP moves backward and places the 3’ end of RNA from DNA-RNA hybrid into the rNTP channel. Transcription pauses until the peeled-back 3’ end is removed by enzymes.

48
Q

What is the role of topoisomerase in transcription?

A

Topoisomerases relaxes the strains introduced into the DNA template caused by RNAP unwinding DNA ahead of the advancing polymerase

49
Q

What are the two types of terminators in E. coli?

A
  1. Intrinsic terminators (rho-independent)

2. Rho-dependent terminators

50
Q

What is the essence of transcriptional termination in bacteria?

A
  1. destabilize RNA-DNA hybrid
  2. pause transcription
  3. make RNAP fall off the template
51
Q

Explain rho-independent termination.

A

RNAP and the transcribed RNA are released when terminator sequences are encountered. RNAP stalls at terminators and causes transcription to pause. This pause promotes formation of transcript secondary structure (hairpin loop) that pulls RNA out of polymerase and/or causes transcription bubble to collapse

52
Q

What are intrinsic terminators?

A

Intrinsic terminators are DNA sequences transcribed into RNA hairpins that stall RNAP. Composed of a stretch of U’s less than 10 nt following the hairpin causes RNA to fall off (because weak A-U bond between DNA-RNA easily detaches transcript from DNA)

53
Q

Explain rho-dependent termination

A
  • Rho is a hexameric ring of identical subunits, hydrolyzes ATP, has RNA-DNA helicase activity
  • Rho binds to rho loading/utilization site (rut site) and stimulates its ATPase activity
  • Rho uses ATP and its helicase activity to move on the transcript in the 5’ to 3’ direction, but maintains interaction with rut site (pulling RNA into a loop)
  • Rho encounters elongation complex releasing RNAP with unknown mechanism.

MBB 331 (3 decks)

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