Transcription Flashcards
List the order in which transcription factors bind to form the eukaryotic PIC.
TFIID, TFIIA, TFIIB, TFIIF + RNA Polymerase II, TFIIE, TFIIH, mediator
TFIID
- 14 subunits
- 3 lobes
- TBP and TAFs
- TAF1 and TAF7 bind to DPE and MTE to form D complex
TBP
- TATA box binding protein
- binds to minor groove and forces open DNA
- highly conserved
- causes DNA to bend 80-90 degrees
TFIIA
- 2 subunits
- replaces TAF1 on TBP binding surface
- TFIIA + TAF1 translocate to TATA box, triggering rearrangement of TFIID
- DA complex
TFIIB
- 1 subunit
- binds to BREu and BREd to stabilize TFIIA+TBP+DNA complex
- DAB complex
BRE
- TFIIB recognition elements
- has flexible domain called B ribbon that hooks RNA polymerase into place on the DNA
TFIIE
- 2 subunits
- forms “clamp” around DNA by binding to RNA polymerase stalk, wrapping around DNA and binding to TFIIF on the other side
TFIIH (+XPB, XPD subunits)
- 10 subunits (most complex GTF) divided into TFIIH core and CAK (cyclin-dependent kinase activating complex)
- catalyzes transition of PIC from closed complex to open complex (enzymatic activity)
- XPB: translocates DNA
- XPD: unwinds DNA
TFIIF
- 2 subunits
- recruits RNA polymerase II to PIC
mediator
- 24 subunits
- 3 lobes (head, middle, tail)
- relays input from enhancers/activators to PIC
RNA polymerase II
- 12 subunits
- transcribes mRNA
- subunit 1 has CTD tail involved in transcription initiation
RNA polymerase holoenzyme
- prokaryotes
- core and sigma factor
- core catalyzes 3’-5’ phosphodiester bonds (polymerization) and consists of 2 alpha, beta, beta prime and omega subunits
- sigma factors recognize promoter
What is each of the 5 subunits of the core enzyme of the RNA polymerase holoenzyme responsible for?
- 2 alpha: recognize UP element
- beta: polymerase activity
- beta prime: binds to DNA
- omega: stability
List the steps of prokaryotic transcription initiation.
- sigma factor attaches to core enzyme
- sigma factor binds to -10 and -35 binding sites
- 2 alpha subunits bind to UP element
- sigma factor loads DNA into beta and beta prime subunits
- complementary bases added to +1 through beta prime entrance channel
- first couple transcripts (~10 nucleotides) aborted due to blocking of exit channel by sigma factor
- elongation begins after sigma factor pushed off
CTD (phosphorylation)
- C-terminal domain of RNA polymerase II subunit 1
- phosphorylated by TFIIH with the help of mediator, occurs at 5th serine in 7 residue sequence YSPTSPS
- involved in hnRNA processing
sigma factor opens the DNA helix from ___ position to ___ position
-10 to +10
RNA polymerase reads DNA _’ to _’, and synthesizes RNA _’ to _’.
- reads DNA 3’ to 5’
- synthesizes RNA 5’ to 3’
Describe intrinsic termination.
- prokaryotic
- mRNA folds into hairpin due to self-complementary G/C rich region
- hairpin pulls adjacent U rich region
- since U rich region is weakly bound to DNA, it is easily pulled off
Describe rho-dependent termination.
- rho protein attaches all 6 subunits on mRNA (it is a homohexamer)
- rho protein adopts lock washer conformation to feed mRNA through its central channel
- rho protein translocates 5’ to 3’ along mRNA and pulls off weak U bonds when it reaches RNA polymerase
How is eukaryotic transcription terminated?
- does not have specific termination signal
- AAUAAA sequence in 3’ UTR of hnRNA signals 3’ trimming and polyadenylation (poly-A tail)
rut (rho utilization site)
- ~70 bases long
- C rich
- where the rho protein initially loads onto mRNA
