Lecture 7 - RNA Pol II promoters and transcription initiation

Question 1

Pre-initiation complex contains _________ factors associated with _________.

Answer 1

General transcription factors (GTFs). RNAP

Question 2

GTFs bind to _________ of genes. Their 2 functions : ….

Answer 2

promoters. 1) Position RNAP (II) at the start site.2) initiate transcr.

Question 3

Transcription initiation can be studied ________ and ______. Difference between the 2 methods

Answer 3

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)

Question 4

First step of transc. initiation

Answer 4

TBP binds to DNA (TATA box) - binds to a minor groove.

Question 5

In ____, TBP is part of ________, which is a complex of __________ and ______________

Answer 5

vivo, TFIID, TBP and 13 other subunits called TAFs (TBP associated factors)

Question 6

2nd step of transcription initiation (after TBP TFIID)

Answer 6

Binding of TFIIA and TFIIB in a complex with TFIID and the tata box.

Question 7

What TFIIB does (2 things)

Answer 7

1) Interacts w/ TBP 2) Binds to sequences of DNA that are 3’ and 5’ of the tata box.

Question 8

3rd step of transcription initiation (after TFIIA TFIIB) (2 *)

Answer 8

Performed complex of TFIIF and Pol II bind the complex and Pol II is positioned at the start site

Question 9

4th step of transcription initiation (after TFIIF pol II)

Answer 9

TFIIE binds and creates a docking site for TFIIH

Question 10

TFIIH, what it is made of

Answer 10

10 subunits, kinase and helicase activity

Question 11

5th step in transcription initiation

Answer 11

One subunit of TFIIH with helicase activity uses ATP to unwind DNA duplex at start site (Pol II forms open complex)

Question 12

After transcription initiation, what other subunit of TFIIH does

Answer 12

other subunit with kinase activity phosphorylates CTD of Pol II

Question 13

After transcription initiation, __________ factors join the complex and _________ factors leave the complex so all _____________ dissociate from the promoter

Answer 13

elongation, initiation, GTFs dissociate from promoter

Question 14

___________ control is the major mechanism for controlling production of a protein. The mechanism is always _______.

Answer 14

Transcription initiation. positive

Question 15

Transcription of a gene can be _________ (_______ mRNA synthesized ) or _______ (_______ times more mRNA synthesized)

Answer 15

repressed (little or no), activated (1000 x more)

Question 16

Reasons for gene regulation in single-celled (unicellular organims) and explanation

Answer 16

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.

Question 17

Reasons for gene regulation in multicellular organisms

Answer 17

to ensure coordination during embryonic development and tissue differentiation

Question 18

4 regulatory sequences in protein-coding genes and what they all interact with

Answer 18

1) BRE (TFIIB recognition element) 2) TATA box 3) Initiator element (Inr) 4) Downstream promoter element (DPE). ALL INTERACT WITH TFIID

Question 19

What is important in regulatory sequences positionning ? Is it necessary to have them all ?

Answer 19

Space conservation. Not all 4 required

Question 20

which regulatory sequence is part of the encoded region

Answer 20

Inr (initiator element) because it incorporates the +1 site.

Question 21

Regulatory sequences will affect ________ positioning when they will interact with __________ and bring __________ to ____________ of the coding region

Answer 21

subunits. TFIID. Pol II. start site

Question 22

Genes that are transcribed at high levels (have strong _______) have a __________ between _____ and ____ nts upstream of the start site

Answer 22

promoters. tata box. 26 and 31.

Question 23

Some genes have an initiator element which includes a ____ at the ___ position and an _____ at the ______ position. No good __________ has been defined

Answer 23

C at -1. A at +1. no good consensus sequence.

Question 24

BRE distance/position

Answer 24

-32 to -37

Question 25

Inr distance/position

Answer 25

-2 to +4

Question 26

DPE distance/position

Answer 26

+28 to +32

Question 27

Which regulatory sequences are associated together or with which promoter

Answer 27

BRE associated with TATA box. DPE associated with Inr and CpG islands

Question 28

Transcription of most protein coding genes in mammals initiates in ___________

Answer 28

CpG islands

Question 29

CpG islands are a stretch of _________ bp that are ______ rich. They are within ________ bp of the start-site region.

Answer 29

20-50. GC. 100.

Question 30

Transcription of genes with CpG islands initiates at _________ _________ sites within a __________ bp region.

Answer 30

multiple alternative sites. 100-1000 bp region

Question 31

Why genes with tatabox are highly transcribed

Answer 31

Because Pol II is always well positioned.

Question 32

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.

Answer 32

phosphate. H bonding

Question 33

What is a cross-linker

Answer 33

substance that does bridges between proteins and stops them in time in protein context. Blocks elongation.

Question 34

2 things that we get with ChIP using antibody to RNAP II.

Answer 34

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

Question 35

First step to ChIP using antibody to Pol II and where it’s done

Answer 35

Treat living tissue or cell with membrane-permeating cross-linker such as formaldehyde. Done in vivo.

Question 36

Other steps of ChIP using antibody to pol II

Answer 36

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

Question 37

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 ________.

Answer 37

orientation. where. divergent. UNIDIRECTIONAL OR BIDIRECTIONAL.

Question 38

What happens in transcription from CpG island where Pol II went in bad direction

Answer 38

Pol II falls off, transcript doesn’t lead to anything and is probably degraded

Question 39

Why 2 transcriptions don’t block each other

Answer 39

Because once transcription is initiated an Pol II leaves the promoter, promoter is clear and available for another pol II.

Question 40

Why you can’t have 2 convergent initiations

Answer 40

Genes usually seperated by long non-coding sequences

Question 41

CpG islands are responsible for transcription of approx. ______ % of genes in mammals.

Answer 41

70

Question 42

_______ of CpG islands can block their binding by ________ factors and ________ the target gene

Answer 42

Methylations. (general) transcription factors. silence

Question 43

3 things promoters do

Answer 43

1) Direct binding of RNAP II to DNA 2) Determine site or transcription initiation 3) Influence frequency of transcription initiation (CpG = lowly transcribed genes)

Question 44

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

Answer 44

100-200. thousands. regulate/control