lecture 2

Question 1

basal transcription alone results in

Answer 1

low/inactive transcription

Question 2

upstream activator sequences (uas) / enhancer elements effects

Answer 2

act as binding site of activator proteins which simulates transcription initiation

Question 3

what are the common sequence UAS/ enhancer elements

Answer 3

GC box, Octamer and CAAT box

Question 4

what transcription factor does GC box bind to

Answer 4

Sp1

Question 5

what factor does octamer bind to

Answer 5

Oct-1

Question 6

what factor does CAAT box bind to

Answer 6

NFY

Question 7

where are enhancer elements located

Answer 7

close to the core promoter (promoter proximal)

Question 8

response elements are

Answer 8

in the enhancer region that are able to bind to transcription factors and regulate transcription of genes

Question 9

response element SRE binds to ?

Answer 9

serum response factor SRF and induced when growth factors are present

Question 10

response element HSE binds to ?

Answer 10

heat shock factor, induced by heat shock

Question 11

response elements bind

Answer 11

factors whose activity is controlled in response to specific stimuli

Question 12

enhancer can be located?

Answer 12

upstream or downstream of the gene they control and at large distances

Question 13

activator protein is the same as

Answer 13

transcription factors

Question 14

activators need 2 domains..

Answer 14

DNA binding domain and activation domain

Question 15

in prokaryotic activators the binding domain and activation domain are

Answer 15

integrated into one region

Question 16

in eukaryotic activators they are

Answer 16

separate and they are linked by a flexible protein domain

Question 17

generally how many binding and activation domains are there in eukaryotic activators

Answer 17

one binding domain and multiple activation domains

Question 18

examples on dna binding domains are

Answer 18

leucine zipper, zinc finger, homeodomain, helix loop helix

Question 19

activation domains are characterised according to their

Answer 19

amino acid sequence

Question 20

activation domains interact with

Answer 20

other proteins in the transcriptional machinery eg TAFs

Question 21

electrophoretic mobility shift assays are used to measure the

Answer 21

ability for an activator to bind to DNA

Question 22

radiolabeled probe DNA and activator is run on

Answer 22

non denaturing acrylamide gel

Question 23

you get two bands if the activator has bound to the DNA and it will of moved down the gel more or less?

Answer 23

less as its a bigger complex

Question 24

transcription assays show

Answer 24

ability for activator to initiate transcription

Question 25

transcription assays require

Answer 25

RNA pol II, GTFs (general transcription factors(TFIIX)), DNA template, radiolabeled rNTPs

Question 26

if the activator works you will see

Answer 26

an radiolabeled RNA transcript in the acrylamide gel

Question 27

in vivo method

Answer 27

one plasmid has activator/TF and one has a reporter gene and a binding site for the protein made, if the protein is made it will bind to the site and expression of the reporter is turned on

Question 28

chromatin immunoprecipitation (ChIP) in vivo method allows us to understand

Answer 28

where transcription factors bind in cells

Question 29

add cross linking agent which

Answer 29

cross links activator protein to DNA

Question 29

add cross linking agent which

Answer 29

cross links activator protein to particular DNA site

Question 30

next you break down the cell and shear DNA into little chunks and then

Answer 30

purify blue sequences by using a specific antibody to the activator

Question 31

reverse cross links by heating

Answer 31

leaving enriched sample of all places n the dna where the activator binds

Question 32

sequence the data using

Answer 32

ChIP-Seq to identify all the binding sites for an activator

Question 33

how do activators work 1. binding of first activator to UAS promotes

Answer 33

binding of a second activator to a different UAS

Question 34

how do activators work 2. binding stimulate recruitment of RNA polymerase, what components of PIC complex so activators interact with

Answer 34

TFIID (via TAFs), TFIIB, mediator (major target)

Question 35

what is mediator

Answer 35

mediator is a large complex of 22 polypeptides, can exist on its own or associated with RNA pol II

Question 36

how many domains to mediator have

Answer 36

3, head, middle and tail

Question 37

mediator subunits interact with

Answer 37

many activators

Question 38

mediator provides a bridge between

Answer 38

activators and RNA pol II

Question 39

mediator - activator interactions aid recruitment of

Answer 39

RNA pol II and therefore enhance PIC formation stimulating transcription

Question 40

the polymerase can stall near to the promoter, this is released by

Answer 40

active activator proteins release stalled RNA pol II

Question 41

heat shock factor binds to activator (HSF transcription factor) and it interacts with RNA pol II

Answer 41

and releases it from the pause