Lecture 3: Mechanism of Transcription

Question 1

What is the difference between the timing and location of transcription & translation of bacteria and eukaryotes?

Answer 1

In bacteria, the timing and location of transcription and translation overlap, where as in eukaryotes it is separated

I.e., Transcription in nucleus, export to ribosomes for translation

Question 2

What are the 3 requirements for transcription?

Answer 2

1. single stranded DNA template
2. All 4 RNA nucleotides
3. DNA dependent RNA Polymerase (holoenzyme consisting of subunits)

Question 3

What is the subunit composition of RNA polymerase

Answer 3

Core enzyme (Alphax2,beta,beta’,(omega)):

• alpha; 40kD each; enzyme assembly and activation
• beta; 155kD; catalytic domain and nucleotide binding
• beta’; 160kD; template binding
• omega; 6kD; unknown function

Holoenzyme (alphax2,beta,beta’,sigma)
- Sigma; various sizes; specificity factor, promoter binding, and open complex formation

Question 4

whats the difference between the core and holoenzyme RNA polymerase

Answer 4

The holoenzyme catalyses RNA synthesis from DNA (just like core enzyme), however this is at the correct targeted locations

Question 5

what are the 4 stages of transcription?

Answer 5

1. template elongation/recognition (RNA polymerase binds and unwinds DNA at the promoter)
2. initiation (chains of 2-9 bases are synthesised and released)
3. elongation (RNA synthesised via base pairing, DNA is unwound as the RNA polymerase moves)
4. termination (RNA polymerase and RNA released at terminator, DNA duplex reforms)

Question 6

What does Ki mean with regards to transcription initiation?

Answer 6

R = RNA polymerase
P = promoter
Ki = equilibrium constant
Ki = RP/(R+P)

Question 7

What does Kii mean with regards to transcription initiation?

Answer 7

The rate of open complex formation

Question 8

What does Kiv mean with regards to transcription initiation?

Answer 8

“promoter clearance”, transition between RPi and RPe

Question 9

How does RNA polymerase binding work?

Answer 9

1. Polymerase binds non-specifically with low affinity looking for promoter
2. sigma subunit recognizes promoter sequence
3. RNA polymerase holoenzyme and promoter form (“CLOSED PROMOTER COMPLEX”) - DNA not unwound. Ki = 10^-6 - 10^-9M
4. polymerase unwinds ~12bp to form (“OPEN PROMOTER COMPLEX”). Kii = 10^-14M

Question 10

What is the function of promoters

Answer 10

attract RNA polymerase to the correct start site so transcription can be initiated

Question 11

How is bioinformatics used to identify and define promoter sites

Answer 11

Identification of many promoter and look for sequence similarities. Used by David Pribnow to define 2 highly conserved sequences -35bp and -10bp upstream to the initiation site

Question 12

What is the relationship between the promoter sequence and the consensus sequence

Answer 12

the more closely related the promoter sequence is to the consensus sequence the stronger the transcription intitiates

Question 13

Where are regions of similarity found for initiation

Answer 13

~10 & 35 bases before start site

Question 14

What is the order of bases in the 10bp-upstream of the start site in prokaryotes. what is it called and whats special about it?

Answer 14

TATAAT - Pribnow box

The bases can differ from promoter to promoter, with different frequencies of each base being present

Question 15

What are the biochemical approaches to defining promoter sites? Explain what it is

Answer 15

DNAse protection.

1. Add purified RNA polymerase to DNA
2. Digest extensively with DNAse (digests all uncovered DNA into nucleotides
3. Isolate and identify the protect region (~60bp in prokaryotes)

Question 16

What is DNAse foot-printing and how can it be used

Answer 16

Finding the promoter region via the DNA protection via an RNA polymerase. It can be used to identify sites where RNA polymerase is in close contact with DNA

Question 17

What is the Genetic approach for defining promoter sites

Answer 17

Mutations in promotor sites can lead to the elimination/reduction of transcriptional initiation.

Strong promoters = sites that are similar to the the consensus sequence
Weak promoters = many differences

I.e., normal protein is produced but at abnormal levels