Transcription + operons Flashcards

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1
Q

2 methods for locating transcriptional start sites

A
  1. S1 nuclease protection (S1 mapping)
    - collect RNA of interest, hybridize to label antisense probe, digest ss (unhybridized), run on gel
  2. primer extension
    - hybridize mRNA w labeled complimentary seg to region close to 3’ end
    - reverse transcribe for cDNA, compare to original DNA seq
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2
Q

other names for nontemplate strand (3)

A

sense
coding
RNA-like

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3
Q

which DNA strand can be template for RNA synthesis

A

both strands

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4
Q

what is a common cofactor in transcription?

A

Mg2+

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5
Q

RNAP characteristics

A
  • DOESNT need a primer
  • promoter sequences are asymmetrical (only transcribe one strand from one promoter)
  • DNA unwound locally only (ATP not needed)
  • product is ssRNA
  • less efficient in proofreading
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6
Q

does reverse transcriptase need a primer?

A

yes

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7
Q

beginning of RNA chain is __, meaning?

A

+1 (first synthesized nucleotide - start of 5’ UTR in mRNA)

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8
Q

upstream vs downstream

A

upstream (-) - from transcription start site to the left (promoter - RNAP binding site)
downstream (+) - from transcription start site to the right

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9
Q

general steps in transcription

A

initiation - binding of RNAP to promoter
- conformational changes of promoter and RNAP
- formation of bubble
- RNAP makes ~10 bp

elongation - enhanced “grip” on template - polymerization
- continue polymerization
- unwinds DNA in front
- a little proofreading
- anneal DNA behind
- dissociate growing chain from template

termination
- diff ways

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10
Q

E. coli RNA pol subunits

A
  • made of 6 subunits (holoenzyme)
  • without sigma subunit = core enzyme
  • 2 alpha subunits - interaction w/ regulatory proteins
  • 2 beta subunits - catalytic subunits
  • 1 omega subunit - needed to restore denatured RNA pol in vitro to functional form
  • 1 sigma (70) subunit - interaction w/ promoter (initiation)
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11
Q

where is consensus TATA box?

A

-10 region

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12
Q

prok promoters vs euk promoters

A

prok promoters: Pribnow box
euk promoters: TATA box

both A-T rich

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13
Q

promoter determines:

A
  • which strand is template
  • starting point for transcription
  • strength of pol binding
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14
Q

transcription steps

A
  • RNAP finds a promoter, sigma70 interacts w/ -10 then -35
  • H-bonds needs to be broken near start site to create open complex; conformational change of RNAP, ATP not needed
  • RNAP makes abortive 10 ntds, S70 slowly released
  • RNAP holoenzyme transcribes
  • S70 fully released
  • core pol continues elongation
  • RNA released
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15
Q

is arabinose operon catabolic or anabolic

A

catabolic

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16
Q

regulatory proteins are mostly

A

homodimers

17
Q

Trp acts as ____, whereas lactose acts as ____

A

corepressor
coinducer

18
Q

AraC regulator - activator upon binding of ____

A

arabinose

19
Q

what if there is too much AraC

A

AraC binds to araO1, prevents more transcription
- RNAP cannot bind to araPc

20
Q

when all 3 operators are occupied in Lac operon, suppression is ____

A

highest!!!

21
Q

repressor could bind to all three of those operators (lac operon) because it binds as a _____

A

tetramer

22
Q
A