Lecture 7 - RNA Pol II promoters and transcription initiation Flashcards
Pre-initiation complex contains _________ factors associated with _________.
General transcription factors (GTFs). RNAP
GTFs bind to _________ of genes. Their 2 functions : ….
promoters. 1) Position RNAP (II) at the start site.2) initiate transcr.
Transcription initiation can be studied ________ and ______. Difference between the 2 methods
in vitro (test tube w/ defined components) or in vivo (genetic techniques). In vitro, add things to DNA template w/ rNTPs and see if transcription happens but in vivo, you may also need an accessory molecule)
First step of transc. initiation
TBP binds to DNA (TATA box) - binds to a minor groove.
In ____, TBP is part of ________, which is a complex of __________ and ______________
vivo, TFIID, TBP and 13 other subunits called TAFs (TBP associated factors)
2nd step of transcription initiation (after TBP TFIID)
Binding of TFIIA and TFIIB in a complex with TFIID and the tata box.
What TFIIB does (2 things)
1) Interacts w/ TBP 2) Binds to sequences of DNA that are 3’ and 5’ of the tata box.
3rd step of transcription initiation (after TFIIA TFIIB) (2 *)
Performed complex of TFIIF and Pol II bind the complex and Pol II is positioned at the start site
4th step of transcription initiation (after TFIIF pol II)
TFIIE binds and creates a docking site for TFIIH
TFIIH, what it is made of
10 subunits, kinase and helicase activity
5th step in transcription initiation
One subunit of TFIIH with helicase activity uses ATP to unwind DNA duplex at start site (Pol II forms open complex)
After transcription initiation, what other subunit of TFIIH does
other subunit with kinase activity phosphorylates CTD of Pol II
After transcription initiation, __________ factors join the complex and _________ factors leave the complex so all _____________ dissociate from the promoter
elongation, initiation, GTFs dissociate from promoter
___________ control is the major mechanism for controlling production of a protein. The mechanism is always _______.
Transcription initiation. positive
Transcription of a gene can be _________ (_______ mRNA synthesized ) or _______ (_______ times more mRNA synthesized)
repressed (little or no), activated (1000 x more)
Reasons for gene regulation in single-celled (unicellular organims) and explanation
To adjust to changes in nutritional and physical environment. Cell produces only proteins required for survival and proliferation under particular env. conditions it lives in.
Reasons for gene regulation in multicellular organisms
to ensure coordination during embryonic development and tissue differentiation
4 regulatory sequences in protein-coding genes and what they all interact with
1) BRE (TFIIB recognition element) 2) TATA box 3) Initiator element (Inr) 4) Downstream promoter element (DPE). ALL INTERACT WITH TFIID
What is important in regulatory sequences positionning ? Is it necessary to have them all ?
Space conservation. Not all 4 required
which regulatory sequence is part of the encoded region
Inr (initiator element) because it incorporates the +1 site.
Regulatory sequences will affect ________ positioning when they will interact with __________ and bring __________ to ____________ of the coding region
subunits. TFIID. Pol II. start site
Genes that are transcribed at high levels (have strong _______) have a __________ between _____ and ____ nts upstream of the start site
promoters. tata box. 26 and 31.
Some genes have an initiator element which includes a ____ at the ___ position and an _____ at the ______ position. No good __________ has been defined
C at -1. A at +1. no good consensus sequence.
BRE distance/position
-32 to -37
Inr distance/position
-2 to +4
DPE distance/position
+28 to +32
Which regulatory sequences are associated together or with which promoter
BRE associated with TATA box. DPE associated with Inr and CpG islands
Transcription of most protein coding genes in mammals initiates in ___________
CpG islands
CpG islands are a stretch of _________ bp that are ______ rich. They are within ________ bp of the start-site region.
20-50. GC. 100.
Transcription of genes with CpG islands initiates at _________ _________ sites within a __________ bp region.
multiple alternative sites. 100-1000 bp region
Why genes with tatabox are highly transcribed
Because Pol II is always well positioned.
In a CpG island, there is a _______ between C and G on the same strand and there is no ___________ between C and G on opposite strands.
phosphate. H bonding
What is a cross-linker
substance that does bridges between proteins and stops them in time in protein context. Blocks elongation.
2 things that we get with ChIP using antibody to RNAP II.
1) We get the genes that are being transcribed
2) We can isolate Pol II and add it to certain chromatin regions in different cell types to see if they are being transcribed
First step to ChIP using antibody to Pol II and where it’s done
Treat living tissue or cell with membrane-permeating cross-linker such as formaldehyde. Done in vivo.
Other steps of ChIP using antibody to pol II
2) Sonicate to shear cellular chromatin to short fragments and add antibody to Pol II
3) Immunoprecipitate to isolate Pol II cross-linked to DNA.
4) Reverse cross-linking, isolate DNA, and subject to massively parallel DNA sequencing
CpG islands give no __________ information to Pol II so it doesn’t know __________. Therefore, ___________ transcription from CpG islands is observed in mammals. However, transcription from a CpG island can be _________ OR ________.
orientation. where. divergent. UNIDIRECTIONAL OR BIDIRECTIONAL.
What happens in transcription from CpG island where Pol II went in bad direction
Pol II falls off, transcript doesn’t lead to anything and is probably degraded
Why 2 transcriptions don’t block each other
Because once transcription is initiated an Pol II leaves the promoter, promoter is clear and available for another pol II.
Why you can’t have 2 convergent initiations
Genes usually seperated by long non-coding sequences
CpG islands are responsible for transcription of approx. ______ % of genes in mammals.
70
_______ of CpG islands can block their binding by ________ factors and ________ the target gene
Methylations. (general) transcription factors. silence
3 things promoters do
1) Direct binding of RNAP II to DNA 2) Determine site or transcription initiation 3) Influence frequency of transcription initiation (CpG = lowly transcribed genes)
Promoter proximal elements are within _________ bp of the promoter and enhancers are long distance elements that can be ________ of kb away from transcription start site. Both ________ transcription
100-200. thousands. regulate/control
