Oct 30 Flashcards
How many transcription factors are needed for transcription prokary vs eukary?
Prokaryotes need only 1 TF, it is the sigma factor. Eukaryotes have many transcription initiation factors
In vitro, what is needed in the promotor for transcription to occur with RNA pol 2 (and what recognizes these spots) , what is this version of the promotor called?
Core promotor is the minimal sequence to get transcription in vitro. Promotor must have BRE (B recognition element) (TF2B (Transcription factor pol 2 B) binds), TATA box (TBP binds (TATA binding protein)), Inr is the initiator and DPE (downstream promotor element) that TF2D binds both of them
What is the layout of the core promotor for Pol 2?
From left to right neg to pos positions, BRE and TATA together, then at -2 to +4 (around zero) have initiator INR that has +1 position, first nuclotide for transcription, then further downstream have DPE
What are some things upstream of the core promotor in eukaryotes for Pol 2 (6)?
Promotor proximal elements, upstream activator sequences enhancers, silencers, boundary elements, insulators
What is the order and function of each element for in vitro transcription initiation in EUKARYOTES?
- TBP binds to TATA box, and recruits TBP associated factors (TAFs) which come next
- TF2B binds to BRE (just upstream of TATA box, they are side by side). It senses and sets the directionality for which way Pol 2 should face
3.TF2F binds to TBP and TF2B that are on the promotor and also brings Pol 2 with it
- TF2F brings TF2E, which recruits TF2H
- TF2H is the largest and most complex TAF, it uses ATP to PO4 C terminal domain (CTD) of Pol 2 and activates it (causing promotor escape)
How does TBP bind to the TATA element?
It has a helix and a B sheet, it bends DNA and binds to the minor groove with the B sheet
How does pre-initiation complex form in vitro?
- TBP binds to TATA box
2.TF2D binds to TBP and promotor
3.TF2A and B bind to TF2D. B sets directionality of transcription
4.TF2F with RNA pol 2 on it binds to the promotor
- C terminal tail of RNA pol 2 binds to the pre nitiation complex
- Still in closed form, then TF2E binds, then H binds (both in front of RNA pol 2 on promotor) which will induce melting of promotor strands with ATP hydrolysis and also PO4’s Pol 2 C terminal tail
- Promotor escape occurs (RNA pol 2 starts moving downstream, E and H get out the way
How does TF2B set the direction of transcription?
It binds to the major groove, forms an assymetric arrangement relative to TBP (which is bending the DNA), so it blocks it, so RNA pol can see which side is not blocked
Which RNA pols (must be eukary as prokary only has 1 RNA pol) have c terminal domain tails?
Just RNA pol 2
Where are upstream activator sequences (UAS’s) located?
They don’t have to be close to promotor, can be very far away (millions of BPs) from promotor
What additional proteins are needed in vivo due to DNA being in chromatin form?
- Activators bind to the DNA
- Mediator complex binds to the pre initiation complex
- The activator proteins bridge DNA and mediator complex together
4.Histone acetyltransferase (removes charge on histone tails) and chromatin remodeler bind to the activator proteins.
Yeast vs Human mediator complexes, what do you see? What subunit is needed for pol 2 transcription in vivo?
See that it is organized into modules (sub-complexes).
2.The complexes between yeast and humans have similar shape and are much larger than RNA pol.
3.Each subunit has a function. Most subunits are well conserved.
Med 17 is needed, it is an example of well conserved subunit
How do you determine what proteins form a complex?
Do immunoprecipitation. You bind antibody to bead, then some modules of the complex will preciptate out and “come down” together. Do SDS page to see what is bound to what. Do this multiple times
What protein helps dissasemble and reassemble nuclosomes so RNA Pol 2 can move freely to do its job?
It is FACT heterodimer. It is in front of RNA pol, and disassembles histones, so now DNA can melt.
Once the DNA is read, then it is reannealed together and FACT resotres the H2A and B dimer so the histones are functional again.
What do elongation factors do?
They stimulate elongation, are recruited to the CTD tail of Pol 2, dependent how much tail is PO4’d.
