initiation, elongation and termination

Question 1

what are the first steps of initiation?

Answer 1

1 - formation of a closed promoter complex: TFIIA, TFIIB and TFIID
2- the transcription factors in the closed complex bind and recruit RNA polymerase II and TFIIF
3 - TFIIE and TFIIH, plus ATP hydrolysis cause formation of open promoter complex

Question 2

what is meant by open promoter complex?

Answer 2

DNA double helix is unwound in the active site

Question 3

how does a tight complex between mediator and the components of RNA polymerase II form?

Answer 3

large surface area

Question 4

which domain is the CTD located on?

Answer 4

RBP1

Question 5

why must the CTD be phosphorylated?

Answer 5

phosphorylated sites serve as recognition points/binding sites for transcription factors

Question 6

how are amino acids phosphorylated?

Answer 6

a basic group gives an electron to the OH of an amino acids (e.g. serine), this OH is now negatively charged and attacks the terminal phosphate on ATP, assisted by Mg2+. this forms Phosphoserine and adenosine diphosphate

Question 7

what is meant by abortive initiation?

Answer 7

when RNA polymerase II terminates after a short distance, small oligonucleotides are produced, but these have poor stability and are degraded. longer hybrids have proper base pairing and are more stable

Question 8

what is promoter clearance?

Answer 8

phosphorylation of serine 5 by TFIIH disrupting the contacts, allowing RNA emergence and RNAP movement

Question 9

in promoter clearance, what position does the RNAP II stop at? what is the function of this?

Answer 9

first nucleosome downstream of the promoter, around 1 position away from RNA splice site. this is suggested as a proofreading function as it is not efficient

Question 10

summarise the events of elongation

Answer 10

1- change of phosphorylation sites on the CTD from serine 5 -> serine 2
2- association of elongation factors with the CTD
3- disassembly and reassembly of nucleosomes
4- methylation on H3K36 triggering deacetylation

Question 11

in elongation, why is phosphorylation changed from S5 of the repeat to S2?

Answer 11

provides a platform for the binding of other components to the C terminal domain

Question 12

in elongation, why are nucleosomes replaced so readily?

Answer 12

regulates gene expression, prevents binding to cryptic promoters

Question 13

why are cryptic promoters a problem?

Answer 13

transcription could start in the middle of the gene, causing RNA-DNA hybrids to be formed

Question 14

what happens to the GTFs in elongation?

Answer 14

TFIIH + TFIIE come off, TFIIB is recycled, and TFIID remains in situ

Question 15

what is the purpose of the ‘wall’ of the RNAP during elongation?

Answer 15

imposes a right-angled turn

Question 16

how are the template and coding strands held apart in elongation? use diagram

Answer 16

forkloops - coding strand is passed over the outside

Question 17

what is the function of the rudder in elongation?

Answer 17

interrupts RNA helix, pushing it out towards the lid and pore

Question 18

what is the function of the zipper in elongation?

Answer 18

ensures template and coding strand align properly

Question 19

how is the RNAP moved along DNA?

Answer 19

ratcheting

Question 20

what is ratcheting?

Answer 20

the process in which the bridge helix is moved out of the site by thermal oscillation, to allow new NTPs to enter it

Question 21

what is the role of the base-tyr stacking intermediate?

Answer 21

prevents 3’ end slipping back into active site

Question 22

outline the process of capping

Answer 22

triphosphatase removes one phosphate from the triphosphate on the end of RNA
->
RNA guanylyltransferase binds the CTD of RBP1, forming a guanosine triphosphate cap
->
RNA guanine-N7 methyltransferase methylates cap

Question 23

what are the functions of capping?

Answer 23

• splicing
• transport
• translation
• stability

Question 24

how are pol II transcripts terminated?

use a diagram

Answer 24

polyadenylation occurs instead of a termination sequence. polyA complex is bound to CTD of RBP1, cuts between AAUAAA and G/U on nascent RNA. polyA tail is added here and exonuclease latches onto the 5’ end in processive fashion, causing RNAP to fall off.