Control of gene expression: transcription

Question 1

is the sense strand of dna coding or non-coding

Answer 1

non coding - transcribed rna has same sequence as synthesised using the template.

Question 2

3 stages of transcription

Answer 2

initiation, elongation, termination

Question 3

what is incorporated and what is generated in transcription

Answer 3

NMP incorporated

pyrophosphate generated

Question 4

NAD+ cap

Answer 4

• stabilises some prokaryotic mRNAs, can be incorporated by RNAP at +1 A, dep on promoter sequence
• NAD structure contains adenine diphosphate
• new mechanism of regulation of prokaryotic gene expression

Question 5

RNAP core vs holoenzyme

Answer 5

holoenzyme - has sigma factor, allows promoter recognition

core enzyme - efficient elongation, multiple start sites

Question 6

RNAP structure

Answer 6

Mg2+ in active site, a cleft between the B and B’ subunits
a CTD can bind DNA
similar to eukaryotic RNAP II, despite there only being high seq identity at the active site

Question 7

identifying promoters - consensus seqs

Answer 7

in silico - align multiple seqs with TSS as reference, degree of match with promoter suggests functional strength. see sequence logo

Question 8

natural promoter mutations

Answer 8

affect mRNA quantity, can be UP or DOWN

Question 9

methods for biochemically mapping DNA-protein interactions - KMnO4

Answer 9

• KMnO4 reacts with unpaired thymine bases, alkali treatment then cleaves at the modified positions - so see where DNA becomes unwound.

Question 10

methods for biochemically mapping DNA-protein interactions - EMSA

Answer 10

electrophophoretic mobility shift assay
- label DNAends with 32P
- incubate with DNA binding protein
- non denaturing gel electrophoresis
- autoradiography
demonstrate sequence specificity with unlabelled competitor DNA titrations - should get decrease in signal complex with WT but not mutant competitor

supershift - test identity of binding protein using an antibody - complex w AB migrates even slower

Question 11

methods for biochemically mapping DNA-protein interactions - modification interference

Answer 11

at what positions does prior chemical modification prevent protein binding on DNA?

• 32P label end of 1 DNA strand
• modify DNA, av 1 per strand
• incubate w protein
• separate bound and unbound DNA (eg gel shift)
• purify DNA, cleave at modified sites
• missing bands = where modification prevents protein binding

Question 12

methods for biochemically mapping DNA-protein interactions - (5)

Answer 12

• DNA footprinting
• EMSA
• modification interference
• KMnO4
• ChIP

Question 13

structure of bacterial promoter

Answer 13

• -35 box
• • 10 box
• +1A
• UP element = AT rich, upstream of -35, recognised by RNAP a subunit CTD - bind via narrowed minor groove, strong promoters
  asymmetry - polarity of transcription
  spacing between - 10 and - 35 important, need correct orientation for RNAP to recognise

Question 14

sigma subunit domains

Answer 14

• domains 2, 4 - recognise -10 and -35 via HTH
• 1.1 = -ve DNA mimic, suppresses inappropriate DNA binding. in free aigma, 1.1 int with 4,prevents DNA binding
  in holoenzyme, 1.1 occupies a DNA binding cleft, reducing non-specific binding - displaced upon promoter binding.

Question 15

events during prokaryotic transcription initiation

Answer 15

promoter DNA binding by holoenzyme
closed binary complex
open binary complex (abortive initiation - binding of 1st NTP low affinity, no stacking)
extension beyond 9 NT…
ternary elongation complex - sigma released

Question 16

initiation vs elongation complex

Answer 16

initiation - seq specific, has sigma subunit

elongation - sequence non specific, stable so processive