Transcription + operons

Question 1

2 methods for locating transcriptional start sites

Answer 1

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

Question 2

other names for nontemplate strand (3)

Answer 2

sense
coding
RNA-like

Question 3

which DNA strand can be template for RNA synthesis

Answer 3

both strands

Question 4

what is a common cofactor in transcription?

Answer 4

Mg2+

Question 5

RNAP characteristics

Answer 5

• 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

Question 6

does reverse transcriptase need a primer?

Answer 6

yes

Question 7

beginning of RNA chain is __, meaning?

Answer 7

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

Question 8

upstream vs downstream

Answer 8

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

Question 9

general steps in transcription

Answer 9

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

Question 10

E. coli RNA pol subunits

Answer 10

• 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)

Question 11

where is consensus TATA box?

Answer 11

-10 region

Question 12

prok promoters vs euk promoters

Answer 12

prok promoters: Pribnow box
euk promoters: TATA box

both A-T rich

Question 13

promoter determines:

Answer 13

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

Question 14

transcription steps

Answer 14

• 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

Question 15

is arabinose operon catabolic or anabolic

Answer 15

catabolic

Question 16

regulatory proteins are mostly

Answer 16

homodimers

Question 17

Trp acts as ____, whereas lactose acts as ____

Answer 17

corepressor
coinducer

Question 18

AraC regulator - activator upon binding of ____

Answer 18

arabinose

Question 19

what if there is too much AraC

Answer 19

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

Question 20

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

Answer 20

highest!!!

Question 21

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

Answer 21

tetramer