basal transcriptional machinery

Question 1

main features of Pol II structure

Answer 1

• DNA enters into a cleft and ‘jaws’ grip the DNA. jaws may open or close
• when DNA gets too close to the active site it’s bound by bridge helix and ‘switch’ regions at base of clamp
  -closure of ‘clamp’ induced by binding of the DNA-RNA hybridge to 3 ‘switch’ regions during elongation= stabilisation
• linear DNA exit is blocked by the ‘wall’ & exits at 90 degree angle
• nucleotides (NTPs) enter via pore below active site

Question 2

eukaryotes have 3 RNA polymerases

Answer 2

• Pol I; transcribes rRNA genes 5, 8, 18, 28S
• Pol II; transcribes all protein encoding genes
• Pol III; transcribes tRNA, RNA and some snRNA genes

Question 3

what does transcription require?

Answer 3

recognition sequences in DNA that lies outside of the transcribed region

Question 4

what is the recognition sequence?

Answer 4

promoter region
- recruits RNA pol to a DNA template
- RNA pol can only move one way

Question 5

what is a TATA box?

Answer 5

DNA sequence that indicates where a genetic sequence can start to be read and decoded
- lies downstream of the start point of the initiatior

Question 6

what is the TATA less core promoter?

Answer 6

a promoter region that has no TATA box

Question 7

what is the consensus sequence?

Answer 7

upstream of the +1 mRNA, TATA box is located -35 ot -25.

Question 8

what is the basal transcriptional machinery?

Answer 8

GTFs (general TFs) and pol II

Question 9

how does the basal transcriptional machinery initiate transcription?

Answer 9

gaining access to DNA which is packed into nucleosomes & higher order chromatin structure

Question 10

what does the largest subunit of Pol II contain?

Answer 10

a C-terminal domain (CTD)

Question 11

what is the CTD?

Answer 11

domain involved in regulaiton of transcription initiaition, elongation and mRNA processing

Question 12

structure of the CTD

Answer 12

52 repeats of a heptapeptide sequence

Question 13

what happens when Pol II is recruited?

Answer 13

recruited to promoter in a hypophosphorylates form and becomes HYPERphosphorylated on serine 2 and 5 during transcription

Question 14

what do inhibitors of CTD do?

Answer 14

inhibit phosphorylation
e.g. DRB = arrest of elongation complexes in vitro and in vivo

Question 15

why is CTD phosphorylation important?

Answer 15

required for promoter clearance (likely to disrupt contacts between pol II and promoter bound factors) and for recruitment of RNA processing complexes e.g. Splicing
+ efficient elongation

Question 16

what is CTD phosphorylated by?

Answer 16

specific cyclin dependant kinases
- can be recruited / activated by transcriptional activators.

Question 17

what makes up the general TFs?

Answer 17

TF II D comprises of 10 TBP + approx. 10 TAFs
- TBP; TAT box associated protein
- TAF; TBP associated factor

Question 18

TFIIB

Answer 18

provides binding surface for Pol II and involved in start-site determination

Question 19

TFIIF

Answer 19

required for accurate initation and recruits TFIIE and TFIIH

Question 20

TFIIH

Answer 20

binds and catalyses ATP dependent unwinding of the start site DNA and phosphorylation of CTD