basal transcriptional machinery Flashcards
main features of Pol II structure
- 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
eukaryotes have 3 RNA polymerases
- 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
what does transcription require?
recognition sequences in DNA that lies outside of the transcribed region
what is the recognition sequence?
promoter region
- recruits RNA pol to a DNA template
- RNA pol can only move one way
what is a TATA box?
DNA sequence that indicates where a genetic sequence can start to be read and decoded
- lies downstream of the start point of the initiatior
what is the TATA less core promoter?
a promoter region that has no TATA box
what is the consensus sequence?
upstream of the +1 mRNA, TATA box is located -35 ot -25.
what is the basal transcriptional machinery?
GTFs (general TFs) and pol II
how does the basal transcriptional machinery initiate transcription?
gaining access to DNA which is packed into nucleosomes & higher order chromatin structure
what does the largest subunit of Pol II contain?
a C-terminal domain (CTD)
what is the CTD?
domain involved in regulaiton of transcription initiaition, elongation and mRNA processing
structure of the CTD
52 repeats of a heptapeptide sequence
what happens when Pol II is recruited?
recruited to promoter in a hypophosphorylates form and becomes HYPERphosphorylated on serine 2 and 5 during transcription
what do inhibitors of CTD do?
inhibit phosphorylation
e.g. DRB = arrest of elongation complexes in vitro and in vivo
why is CTD phosphorylation important?
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
what is CTD phosphorylated by?
specific cyclin dependant kinases
- can be recruited / activated by transcriptional activators.
what makes up the general TFs?
TF II D comprises of 10 TBP + approx. 10 TAFs
- TBP; TAT box associated protein
- TAF; TBP associated factor
TFIIB
provides binding surface for Pol II and involved in start-site determination
TFIIF
required for accurate initation and recruits TFIIE and TFIIH
TFIIH
binds and catalyses ATP dependent unwinding of the start site DNA and phosphorylation of CTD
