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Molecular Genetics > Prokaryotic Transcription 1 > Flashcards

Prokaryotic Transcription 1 Flashcards

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1
Q

coding strand

A

has the same sequence as the RNA

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2
Q

transcription unit

A
  • from a single promoter
  • may encode more than a single gene (polycistronic)
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3
Q

what is the rate of transcription?

A

50 nucleotides as opposed to 833 in replication

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4
Q

what is the stream?

A
  • the flow of RNA polymerase
  • downstream is the direction of synthesis
  • upstream is the opposing, the promoter is what determines synthesis
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5
Q

transcription bubble

A
  • transcription occurs by base pairing in a bubble of unpaired DNA
  • the region of DNA that opens so the polymerase can read he strand
  • the length of the bubble is 12-14 basepairs
  • the length of the RNA-DNA hybrid is 8-9 nt
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6
Q

RNA-DNA hybrid

A
  • adds nucleotides to the 5’ end
  • short portion that remains as a hybrid
  • RNA polymerase separates the new product from the template
  • as the transcription bubble moves along the DNA template, the RNA is displaced from the RNA-DNA hybrid
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7
Q

what direction is RNA synthesized in?

A
  • synthesized from 5’ - 3’
  • the incoming nt loses the gamma and beta phosphate groups, leaving the alpha phosphate
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8
Q

what is the length of DNA-DNA at room temperature?

A
  • short pieces of DNA easily denature
  • 11 base pairs is on the border of what is stable at ambient temperatures
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9
Q

what is the rate of transcription?

A

40-50 nt per second, which is about the same rate as translation
- transcription is much slower than DNA synthesis

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10
Q

what is the rate of translation?

A

15 amino acids per second/ 45 nt of RNA per second
- as RNA emerges from the RNA polymerase, it is immediately used as substrate for ribosomes
- translation cannot be faster than transcription otherwise it would interfere with the RNA polymerase

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11
Q

how do ribosomes bind to RNA?

A
  • the ribosomes scans at a ribosome binding site looking for AUG
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12
Q

not long after making mRNA the cell starts to degrade , why?

A

the cell is degraded by RNases 1.5-3 minutes after mRNA is made. This is because the cell has a short life already and the mRNA wouldn’t be needed for long

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13
Q

what are the proposed mechanisms for how RNA polymerase finds a promoter

A
  • the observed rate at which RNA polymerase finds a promoter sequence is too fast to be explained by simple diffusion
  • sliding
  • intersegment transfer
  • intrasegment transfer (hopping)
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14
Q

how many molecules are in an E. coli cell

A

13,000 molecules

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15
Q

where is the core RNA polymerase before transcription begins?

A

core RNA polymerase is stored at nonspecific DNA site before sigma binds. Core enzyme has a high intrinsic affinity for non-promoter DNA, which is increased by the presence of nascent RNA
- but its affinity for loose binding sites (non-promoter DNA) is too high to allow the enzyme to distinguish promoters efficiently from other sequences

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16
Q

describe the role of sigma

A
  • when sigma subunit binds to the core of the RNA polymerase, you get a holoenzyme
  • with sigma, the polymerase positive groove in the enzyme opens and loses its affinity for DNA
  • when it binds to a region of DNA that is a promoter, it increases its affinity to the non specific DNA site 1000 fold
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17
Q

true/false: sigma decreases the affinity of the core for DNA

A
  • cannot be answered
  • causes 10,000 fold lose in affinity for nonspecific DNA
  • causes 1000 fold increase in affinity for promoter DNA
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18
Q

what happens when the holoenzyme finds a promoter?

A
  • forms a closed promoter complex that is reversable
  • if there is not good contact, then the complex can disassociate
  • only step considered to be reversible
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19
Q

closed promoter

A

DNA strands have not been melted

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20
Q

open promoter

A
  • melts the two strands in order to read the template strand
  • mediated by sigma subunit with a special domain
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21
Q

once the open promoter forms, what occurs in the holoenzyme?

A
  • RNA polymerase does not need a primer
  • RNA polymerase core tries to transcribe but often has 100-200 false starts
  • once it has the first nucleotides, the subunit is still attached to the promoter and prevents transcription
  • sigma subunit is stick in the RNA exit pore
  • DNA scrunching of 9-8 nucleotides occurs until the sigma unit releases
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22
Q

ternary complex

A
  • most stable
  • protein, DNA, and RNA
  • formed after the first phosphodiester bond is formed
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23
Q

what percentage of RNA polymerase is actually engaged in elongation of transcription? what percentage is used in storage?

A

25% / 50%

24
Q

what happens to DNA during transcription?

A

as a consequence of transcription, DNA in front of the polymerase is overwound, and the DNA behind is underwound
- two compensate, gyrase in fron to introduce negatve supercoils and topisomerase 1 and 4 are trailing to relax the supercoils

25
Q

what causes most of the supercoiling in DNA?

A

probably results from transcription

26
Q

what are the three stages of transcription?

A
  • initiation (template recognition, closed promoter complex, open promoter complex, and abortive cycling)
  • elongation
  • termination
27
Q

what results in the formation of the open promoter?

A
  • irreversible and fast
  • involves the melting of the DNA assisted by sigma
28
Q

what is the fastest promoter clearance time? which promoter?

A
  • ribosomal promoter is the fastest at 1 second
  • still has false starts
29
Q

what part of the transcript is the most unstable and what is the half life for eukaryotes and prokaryotes?

A
  • primary transcript is the most unstable
  • in prokaryotes it is degraded in 1-3.5 mins or cleaved to give mature products such as rRNA or tRNA
  • in eukaryotes it is modified at the ends (mRNA 7mG CAP at 5’ end and polyA tail at 3’ end) or cleaved to give mature products (splicing or rRNA/tRNA)
30
Q

how many human cells die everyday? how many blood cells die every second?

A

330 billion / 5 million

31
Q

how long is the RNA/DNA hybrid at the active site of transcription?

A

9-8 nt

32
Q

Phage T7 RNA polymerase

A
  • T3 and T7 phage RNA polymerases are single polypeptides - only on unit, not multiple subunits
  • they have minimal activities in recognizing a small number of phage promoters. uses a specificity loop to recognize the promoter
  • contains thumbs and fingers
  • promoter is very short (only 6-7 nucleotides)
  • the rate of synthesis is greater than bacterial RNA polymerase at about 200 nucleotides per second
33
Q

what do the crystal structure of T7 RNA polymerases identify in DNA?

A
  • the DNA binding region
  • the active site
34
Q

where does the specificity loop bind to?

A

the T7 RNA polymerase has a specificity loop that binds to positions -7 to -11 of the promoter while positions -1 to -10 enter the active site
- the enzyme holds 10 bp upstream and 10 bp downstream from the transcription bubble
- the bases of the template strand are flipped out of the helix to be read

35
Q

how large is the groove in bacterial RNA polymerase

A

25 angstroms wide and could hold 16 nt of DNA
- groove is positively charged but most of the enzyme is negatively charged

36
Q

what is nascent RNA?

A

RNA that is in the process of being formed

37
Q

Label all the parts

A

1 = beta prime
2 = main channel
3 = beta
4 = alpha 1
5 = alpha 2
6 = omega

38
Q

what happens to RNA polymerase before elongation?

A
  • it goes trough several conformational changes
  • closed promoter = -55/+1
  • open promoter = -55/+20 (DNA melted)
  • ternary complex = (-30/+20)
39
Q

how many magnesium ions do all RNA polymerases have?

A

2 at the active site

40
Q

domain 3.2

A
  • protrudes into the RNA exit pore and assists in the first nucleotide interaction
41
Q

domain 2.3

A

triggers melting

42
Q

domain 1.1

A

displaced by DNA and lies in the exit pore channel

43
Q

clamp

A

on each side of the DNA and forms a channel

44
Q

bridge

A

used to help incoming nucleotides in the channel

45
Q

rudder

A

ensures RNA/DNA region is not longer than 8 nt in length

46
Q

wall

A
  • backside of the active site and DNA makes contact with it
  • assists in melting and bends the DNA to facilitate the opening of the DNA
  • open promoter complex
47
Q

why does DNA bend when at the active site?

A
  • helps flip the RNA away from the template strand
  • the rigid structure of the double stranded DNA becomes flexible when the strands melt
  • the rudder limits the length of the RNA/DNA hybrid
48
Q

how does the enzyme bind the promoter without it hitting the wall?

A

a large conformational change must take place
- the clamp is initially out of position and repositions to keep the DNA in the active site after the DNA has melted

49
Q

how does the RNA polymerase combat keeping proper positioning of the template and mascent transcript?

A

a protein bridge changes conformation to control the entry of nucleotides to the active site
- maintains contact with the growing strand during translocation step but snaps back once the bonds have formed

50
Q

which domain of RNA polymerase prevents the RNA/DNA hybrid from getting too long?

A

rudder/lid

51
Q

beta and beta prime

A

form walls of the active site
- catalytic center

52
Q

alpha subunits

A
  • controls the affinity of the holoenzyme for the promoter
  • regulate frequency of initiation
  • has flexible linkers
  • carboxyl terminal of alpha interacts with the DNA and sees the promoter sequence in the minor groove
53
Q

how large is the RNA core? with the sigma subunit?

A
  • RNA core = 460 kD
  • w/ sigma = 530
54
Q

which subunit is required for promoter specificity?

A

sigma

55
Q

what are the three roles of alpha?

A
  • contacts DNA directly
  • contact transcription regulatory proteins
56
Q

what are the two outcomes of scrunching?

A
  • sigma leaves or RNA leaves

Molecular Genetics (28 decks)

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