RNA Polymerase And Transcription

Question 1

at what level is gene expression controlled

Answer 1

transcription level, turning genes on and off

Question 2

what are the phases of transcription

Answer 2

initiation, elongation and termination

Question 3

what is a holoenzyme

Answer 3

an RNA polymerase enzyme which is associated with a sigma factor

Question 4

explain closed complex

Answer 4

the reversible first stage on initiation. the sigma is bound to RNAP which allows location of the promoter and the DNA duplex is still closed

Question 5

explain open complex

Answer 5

the irreversible second stage of initiation where mRNA transcription begins and DNA unwinds

Question 6

explain abortive initiation

Answer 6

the third stage of initiation. around 10bp are transcribed but it can’t go further as the sigma factor is still bound to the promoter

Question 7

explain processive initiation

Answer 7

the final stage of initiation. sigma is ejected which allows polymerase to form a ‘crab claw’ on DNA and elongate mRNA, locating it in the exit channel

Question 8

what is RNAP composed of

Answer 8

two alpha subunits, a beta, a beta prime, an omega, and this is associated with a sigma factor

Question 9

how does sigma bind to the primer

Answer 9

it has two projecting lobes- sigma 2 binds to -10 and sigma 4 binds to -35

Question 10

where does the DNA break first in the open complex

Answer 10

at the -10 region, it is rich in AT and therefore the bonds are weaker

Question 11

why is sigma ejected

Answer 11

two reasons- it prevents the elongation of mRNA beyond 10bps and and it blocks the exit channel

Question 12

where does recognition of the promoter occur

Answer 12

the -35 region of the promoter and the sigma 4 lobe via direct or indirect readout

Question 13

what does 6S RNS do

Answer 13

it accumulates in the stationary phase and mimics the open complex, preventing transcription. it dissociates with the increase of NTPs

Question 14

explain catalysis

Answer 14

the previous nucleotide is translocated to the i region to free up the i+1 region. the new NTP is hydrogen bonded to the exposed base of the DNA template, then a phosphodiester bond to the previous nucleotide forms, releasing the resultant PPi

Question 15

how is transcriptional accuracy ensured

Answer 15

two Mg2+ ions and the trigger loop/ helix are involves. one Mg2+ is associated with the core and one with the incoming NTP, if it is the right one the trigger helix pushed it into place next to the first ion, allowing catalysis to happen

Question 16

what interrupts transcription

Answer 16

antibiotics or DNA templates with gaps in can cause stalling or arrest

Question 17

what are the two types of termination

Answer 17

intrinsic (the sequence forms hairpin loops which open the exit) and Rho dependent (Rho proteins disengage the RNAP from the mRNA

Question 18

what are the two important sigma variations

Answer 18

sigma 70, expressed in exponential phase, and sigma 38, expressed in stationary phase

Question 19

how is the expression of sigma 38 changed

Answer 19

molecules can upregulate it (eg ppGpp) or downregulate it (eg cyclic AMP), they both alter expression of rpoS gene which encodes sigma 38

Question 20

what is ClpP

Answer 20

a protease responsible for sigma 38 degradation- activity reduces in stationary phase to allow competition with sigma 70

Question 21

what is the stringent response

Answer 21

reaction to amino acid starvation- works by inhibiting transcription of translation machinery eg tRNA, and by enhancing transcription of amino acid biosynthesis genes

Question 22

how does control of genes work

Answer 22

regulation is intrinsic to the DNA of the promoter region of the controlled gene- both negative or positive regulation

Question 23

describe negative control

Answer 23

the CC region of the G rich discriminator in the non- template strand binds to ppGpp, taking with it a Mg2+ ion which prevents catalysis and also tightens the RNAP-DNA complex

Question 24

describe positive control

Answer 24

the discriminator in the non- template strand is AT rich so ppGpp can’t bind, catalysis can’t be stopped and the RNAP-DNA complex is loosened. it also upregulates transcription to produce sigma 38, stimulating transcription of more stress genes

Question 25

what is DksA

Answer 25

a protein which binds to the RNAP on the secondary channel and traps the trigger loop, preventing catalysis

Question 26

how is DNA repaired

Answer 26

ppGpp repairs DNA by base excision repair

Question 27

what is phase variation

Answer 27

changing whether the expression of a gene is on or off depending on which direction a promoter is flipped

Question 28

what type of promoters does phase variation affect

Answer 28

divergent promoters which alternate in direction

Question 29

explain phase variation in Salmonella

Answer 29

fliC encodes H2 flagellin and fliB encodes H1- one on either side of the promoter and inverting region. rh1 encodes a repressor which prevents the transcription of H1 when promotion is in the direction of H2

Question 30

what is the structure of the inverting region

Answer 30

it is a Hin gene with two hix sites, one on either side, and inversion is stimulated by the binding of proteins Fis and HU

Question 31

why is phase variation important

Answer 31

it is significant in the immune response and environmental response (eg temperature change)