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LF104: Molecules, Cells And Organisms > 11 - Bacterial Transcription > Flashcards

11 - Bacterial Transcription Flashcards

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1
Q

which strands are ‘sense’ and ‘nonsense’ in bacterial transcription

A

5’ - 3’ - non-template ‘sense’ strand

3’ - 5’ - template strand ‘antisense’ strand

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2
Q

Difference in base pairing in RNA synthesis in bacteria and DNA synthesis in eukaryotes

A

RNA synthesis in bacteria contains Uracil as a base instead of Thymine
- othewise, follows same base-pairing rules as DNA

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3
Q

Why is uracil not found in DNA

A

Any uracil produced by spontaneous deamination of cytosine can cause mutations in daughter strands
- so is removed

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4
Q

How is uracil produced as a base in DNA
- why is this bad?

A

Cytosine bases in DNA replication can undergo spontaneous deamination to produce uracil
- can lead to mutations if this occurs, as a mutant daughter strand

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5
Q

How is uracil removed as a base in DNA replication, and how is DNA fixed after

A
  • U is removed by uracil-DNA glycolsylase
  • generates an abasic site
  • absasic site is removed and repaired by DNA polymerases
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6
Q

3 major classes of bacterial RNA and functions

A

mRNA - encodes proteins
rRNA - constituents of ribosomes: role in translation and protein synthesis
tRNA - adaptors between mRNA and amino acids: role in protein synthesis

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7
Q

RNA Polymerase function in E. coli
- how differs from eukaryotes

A

synthesises the 3 major classes of bacterial RNA (mRNA, rRNA, tRNA)
- in eukaryotes, there is separate RNA polymerase for each class

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8
Q

advantage of bacteria having no nucleus in transcription and translation

A

allows transcription and translation to occur simultaneously

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9
Q

bacterial transcription unit

A

often an operon

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10
Q

Function of different parts of operon in bacterial transcription

A

5’ promoter - attracts and binds RNA polymerase - expect an operator
Protein coding (transcribed) sequences - often multiple genes (polycistronic) - part of operon
3’ terminator region - signals stop point for transcription

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11
Q

structure of Bacterial RNA polymerase
- how to convert to holoenzyme

A

bacterial core RNA polymerase made of multiple subunits:

  • alpha
  • Beta
  • Beta’
  • w (omega) subunits
  • in ratio of 2:1:1:1
  • addition of a sigma subunit converts enzyme to a holoenzyme
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12
Q

RNA Polymerase binding to DNA at promoter sequences

A
  • core RNA polymerase binds DNA non-specifically and can slide
  • sigma subunit binds to core polymerase to produce holoenzyme
  • directs holoenzyme to a gene promoter to transcribe DNA
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13
Q

Method to isolate promoter region in operon in bacteria
- what this method forms basis for

A
  • bind RNA polymerase holoenzyme to DNA in vitro
  • Add nuclease
  • DNA is degraded, except for stretch bound to polymerase, which is protected
  • forms basis of DNA footprinting technique used to identify promoters
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14
Q

regions on bacterial transcription units that are protected by RNA polymerase

A
  • 2 promoter regions
  • both near start of transcription (-10 sequence and -35 sequence from start)
  • both sites have conserved bases (e.g. TTGACA) and are shown in most promoter regions
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15
Q

directionality of promoter sequence

A
  • asymmetry of promoter region provides directionality
  1. -10 and -35 start sequencea are defined on SENSE (non-template, non-transcribed) strand
  2. RNA built in 5’ to 3’ direction
    - new nucleotides added at 3’ end
    - using antisense strand as a template (GC, AU)
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16
Q

3 stages of transcription
- brief summary of each

A
  • initiation - RNA polymerase holoenzyme binds promoter, opens DNA double helix, and starts to transcribe
  • elongation - sigma subunit disengages from holoeznyme, core enzyme continues to make new RNA
  • termination - RNA polymerase core enzyme dissociates from DNA, transcription halts
17
Q

Transcription of bacterial DNA - initiation mechanism

A
  • core polymerase consists of 2 alpha, 1 B, 1 B’ and 1 omega subunit
  • the core DNA polymerase binds DNA non-specifically and can slide
  • An alpha subunit binds to the core polymerase and directs the polymerase holoenzyme to a promoter
  • holoenzyme binds to the -10 and -35 regions
  • there are multiple alpha factors, which can bind to different promoter regions - specificity

Scrunching, abortive initiation and success:
- polymerase pulls downstream DNA towards itself
- scrunching the DNA
- until success, where -10 region is opened
- this converts closed promoter complex to an open promoter complex - does not require ATP hydrolysis (unlike DNA helicase)

  • 12 to 15 bp are unwound from within the -10 region to position to +2 or +3 - transcriptional start site is exposed
  • RNA polymerase makes RNA copy from the template strand using bp rules (CG, UA)
  • RNA polymerase does not require a primer (unlike DNA Pol)
  • After around 10 nucleotides of RNA synthesis, alpha factor is now exposed and disengages
  • RNA polymerase can now elongate the new RNA - transcriptional elongation now occurs
18
Q

Transcription - elongation mechanism

A
  • transcription ‘bubble’ forms for RNA Pol function
  • during elongation, RNA polymerase is highly processive of DNA strands - DNA is processed quickly through the RNA pol (polymerase) core to produce RNA

proofreading:
- if RNA Pol mis-incorporates a ribonucleotide
- it hesitates and then back-tracks to remove the nucleotide
- then continues transcrip
- Error rate: 1 mistake every 10^4 - 10^5 nucleotides, even with proof-reading

19
Q

Transcription - termination mechanism

A

2 mechanisms:
- Rho (p)-independent - terminator sequence in RNA is recognised
- Rho (p)-dependent - requires Rho (p) protein to break the RNA:DNA duplex in transcription

  • In both cases, the functioning signals are recognised not in the DNA template, but in the newly synthesised RNA.

Rho (p)-independent:
- DNA encodes STOP signals for transcription
- simplest encoding is a GC rich sequence, followed by a T rich sequence
- transcription terminates in this run of Ts or just after them

Rho (p)-dependent termination:
- Rho (p) protein is a hexameric helicase that binds a C-rich, G-poor sequence in RNA
- uses its helicase activity to chase RNA pol
- it then caches
- this disrupts the DNA:RNA hybrid helix
- releasing the RNA

20
Q

what provides directionality to bacterial DNA

A

asymmetry of the promoter sequence

21
Q

how specificity provided for different promoters in bacterial transcription

A
  • different alpha subunits and factors recognise different promoters and provide specificity
22
Q

Rate of transcriptional elongation in bacteria compared to eukaryotes (with DNA Pol)

A

bacteria have slow elongation compared to DNA Pol in eukaryotes

23
Q

Rate of transcriptional elongation in bacteria (RNA Pol) compared to eukaryotes (with DNA Pol)

A

bacteria have slow elongation in RNA Pol compared to DNA Pol in eukaryotes

24
Q

two transcriptional termination mechanisms in prokaryotes

A

Rho (p)-independent - a terminator sequence in RNA is recognised
Rho (p)-dependent - requires Rho (p) protein to break the RNA:DNA duplex in transcriptional bubble

25
Q

why such a high error rate in prokaryotic RNA transcription elongation

A
  • DNA errors are transmitted to progeny cells: they are stringently repaired.
  • RNA errors mean that some transcripts may be mutated, but the majority are not.
  • If the transcript encodes a protein, them most of that protein will be fine, but a small subpopulation may be mutant – and can probably be tolerated.
26
Q

prokaryotic transcription inhibition

A
  • Rifampicin is an inhibitor
  • inhibits RNA Pol by binding tightly to RNA exit channel
  • so affects initiation (but nor RNA Pol at elongation stage)
27
Q

RNA Pol bending DNA duplex use

A

allows the duplex to be opened up more easily

LF104: Molecules, Cells And Organisms (25 decks)

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