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Genes, Genomes and Biomedical Implications > Prokaryotic Transcription > Flashcards

Prokaryotic Transcription Flashcards

1
Q

What is RNAP?

A

an enzyme that synthesises RNA using one strand of duplex DNA as a template in the 5’ to 3’ direction

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2
Q

What is the coding strand?

A

the DNA strand that has the same sequence as the mRNA

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3
Q

What is a transcription unit?

A

a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator

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4
Q

What is a promoter?

A

a region of DNA where RNAP binds to initiate transcription

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5
Q

What is a terminator?

A

a sequence of DNA that causes RNAP to terminate transcription

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6
Q

What is the start point (+1)?

A

the position on DNA corresponding to the first base incorporated into RNA (one nt downstream of the promoter)

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7
Q

What is the length of the transcription bubble?

A

~12 to 14 bp

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8
Q

How big is the DNA-RNA hybrid within the transcription bubble?

A

~8 to 9 bp

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9
Q

What happens as the transcription bubble progresses?

A

the DNA duplex reforms behind it, displacing the RNA in the form of a single polynucleotide chain

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10
Q

What is the transcriptional and DNA replication rate respectively?

A
  • transcription = ~40-50 nt/s
  • DNA replication = ~800 bp/s
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11
Q

What is nascent RNA?

A

an RNA chain that is still being synthesised, so that its 3’ end is paired with DNA where RNAP is elongating

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12
Q

What are the 3 stages of transcription?

A
  1. initiation
  2. elongation
  3. termination
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13
Q

How is the closed complex formed?

A

RNAP binding to the promoter on the DNA

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14
Q

How is the open complex formed?

A

σ factor binds to RNAP and the holoenzyme opens the DNA duplex to form the transcription bubble

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15
Q

What happens during elongation?

A

the transcription bubble moves along DNA and the RNA chain is extended in the 5’→3’ direction

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16
Q

What happens at termination?

A

the DNA duplex reforms and RNAP dissociates at a terminator

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17
Q

What is a holoenzyme?

A

the form of RNAP that initiates transcription

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18
Q

What are the 5 subunits of RNAP?

A
  • 2α
  • β
  • β′
  • ω
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19
Q

What is the catalytic domain of RNAP made up of?

A

the β and β’ subunits (the 2 largest subunits)

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20
Q

What do the α subunits of RNAP do?

A

serve as a scaffold for assembly and interacts with the promoter and some regulatory factors through its C-terminal domain (CTD)

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21
Q

What does the ω subunit of RNAP do?

A

promoter specificity

22
Q

What is σ factor involved in?

A

the melting (unwinding) reaction; it recognises the promoter and binds to the core RNAP

23
Q

What is the ternary complex?

A

the complex involved in initiation of transcription that consists of RNAP, DNA and nascent RNA

24
Q

What does the initiation complex span?

A

-55 to + 20

25
Q

When is σ factor released from the initiation complex?

A

when initiation succeeds and the nascent RNA chains reach ~10 bases in length

26
Q

What does RNAP transition to after initiation?

A

elongation ternary complex of core RNAP-DNA-nascent RNA

27
Q

How big is the elongation complex?

A

30-40 bases

28
Q

What happens after σ factor dissociates?

A

the core enzyme contracts to -35 then after moving a few base pairs, it becomes more compactly organised into the general elongation complex

29
Q

What does σ factor do upon interaction with the core enzyme?

A

change its structure to expose its DNA-binding domains

30
Q

Which σ regions recognise the -10 and -35 hexamers respectively?

A
  • -10 = 2.5 region
  • -35 = 4.2 region
31
Q

What does the N-terminus of σ factor do?

A

block the DNA-binding regions from binding to DNA

32
Q

How many σ factors does E. coli have and what do they cause?

A

7, each of which causes RNAP to initiate at a set of promoters defined by specific –35 and –10 sequences

33
Q

What is σ 70 used for?

A

general transcription (recognises one set of promoters)

34
Q

When is a cascade of σ factors created?

A

when one sigma factor is required to transcribe the gene coding for the next sigma factor

35
Q

What does termination require?

A

recognition of the terminator sequence in DNA and the formation of a hairpin structure in the RNA product

36
Q

What does antitermination cause?

A

the enzyme to continue transcription past the terminator sequence, readthrough

37
Q

What is intrinsic termination?

A

when the core enzyme can terminate at certain sites in the absence of any other factors

38
Q

What does intrinsic termination consist of?

A

a GC-rich hairpin in the RNA product followed by a U-rich region where termination occurs

39
Q

What must happen to form the hairpin loop?

A

a palindromic sequence must be formed

40
Q

What is Rho factor?

A

a terminator protein that binds to a rut site on a nascent RNA and translocates along RNA until it reaches the RNA–DNA hybrid in RNAP

41
Q

What is Rut?

A

the Rho utilisation site, the sequence of RNA that is recognised by the Rho termination factor

42
Q

What is coupled transcription/translation in bacteria?

A

when ribosomes begin translating mRNA before transcription is complete

43
Q

Describe bacterial mRNA

A

unstable with a half-life of only a few minutes

44
Q

What is the 5’ UTR?

A

untranslated sequence upstream from the coding region of an mRNA

45
Q

What is the 3’ UTR?

A

untranslated sequence downstream from the coding region of an mRNA

46
Q

What is monocistronic mRNA?

A

mRNA that encodes one protein

47
Q

What are polyribosomes?

A

a cluster of ribosomes, bound to a mRNA molecule

48
Q

What does DNA footprinting do?

A

identify DNA-binding sites for proteins by their protection against nicking

49
Q

What happens in DNA footprinting?

A

a bound DNA-binding protein blocks the phosphodiester bonds from attack (nuclease or chemicals), revealing the protein precise recognition site as a protected zone, or footprint

50
Q

What is EMSA used for?

A

to study if and how a particular protein interacts with DNA

51
Q

What happens in EMSA?

A
  1. when a protein binds to DNA, it will cause DNA/protein complex to move slowly through the gel
  2. the larger the bound protein, the greater the retardation of the DNA molecule
  3. gel-mobility shift assay traces the amounts of a sequence- specific DNA binding protein

Genes, Genomes and Biomedical Implications (15 decks)

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