Lecture 2 - Activators & Mechanisms of Transcriptional Activation

Question 1

what happens in the absence of an activator?

Answer 1

the PIC is largely inefficient

Question 2

what are the 2 classes of UAS/enhancer elements?

Answer 2

common sequence elements

response elements

Question 3

what are common sequence elements?

Answer 3

often located close to the core promoter - proximal elements

binds activators that are relatively abundant and always switched on

examples:
• GC box
• octamer
• CAAT box

Question 4

what are response elements?

Answer 4

allow the promoter to respond to a particular signal

examples:
• SRE: binds serum response factor (SRF)
• HSE: binds heat shock factor

Question 5

what do these elements do?

Answer 5

combination of elements dictates when and what level a gene is transcribed

Question 6

location of enhancers

Answer 6

enhancers work irrespective of their location

if you reverse the orientation or if its downstream it will still work

they can be brought closer by the looping of DNA

Question 7

what are the 2 domains in eukaryotic activators?

Answer 7

• 1 binds to the DNA site
• 1 binds to the activation domain

separable modules

there is always 1 binding domain but there can be mire than 1 activation domain

Question 8

DNA binding domains

Answer 8

well defined structures

if you take apart any part of the structure they don’t tend to work

Question 9

activation domains

Answer 9

lack sequence conservation and structural information

unstructured until they react with their target protein

contain multiple short segments that work together in an additive fashion

Question 10

methods for the analysis of activators

Answer 10

reporter assays

in vivo approaches

Question 11

what are in vivo approaches for the analysis of activators?

Answer 11

ways of measuring where an activator binds

• DNA foot printing
• electrophoretic mobility shift assays (gel shift)
• transcription assays

Question 12

what are electrophoretic mobility shift assays (gel shift)?

Answer 12

measures the ability of an activator to bind a specific sequence

1. radiolabel probe DNA and run it on a non-denaturing acrylamide gel
2. if protein binds it forms a complex which shifts the probe DNA - gel shift

probe is usually always in excess so is always there in results

Question 13

what are in-vivo transcription assays?

Answer 13

measures ability of protein to activate transcription

RNA pol II + GTFs + DNA template + radiolabelled rNTPs

• put them all in a test tube
• get a radiolabellled transcript
• will give a band in the gel
• tells you activator has activated transcription

Question 14

what does an in-vivo transcription assay require?

Answer 14

the activator to both have a functional DNA binding domain (DBD) and a functional activation domain (AD)

Question 15

what is chromatin immunoprecipitation (ChIP)?

Answer 15

used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo

powerful technique

Question 16

how does chromatin immunoprecipitation work?

Answer 16

1. add cross linking agent to glue all proteins in place
2. can then isolate chromatin and cut DNA up
3. use an antibody specific to protein of interest
4. selectively purifies all protein and its binding sites
5. protein is still cross-linked to DNA - reverse cross link and digest protein with enzyme
6. have purified sample left

Question 17

how can you analyse the purified sample left after chromatin immunoprecipitation?

Answer 17

PCR

microarray

sequencing

Question 18

what are microarrays?

Answer 18

show relative levels of gene expression in a cell line of interest and a control so we can see the effect of transcription factors in different conditions

looks like a microscope slide but has thousands of probes present

Question 19

what are the probes in microarrays?

Answer 19

each one is an oligonucleotide specific to a particular gene in the genome

Question 20

process of microarrays

Answer 20

1. samples prepared - control and experiment
2. isolate mRNA
3. produce cDNA
4. label 2 experiments with different dyes
5. mix them together and hybridise to the microarray slide
6. probes hybridise to the complimentary probes on the microarray

Question 21

what do the results from a microarray show?

Answer 21

usually:
• red = gene is unregulated 
• green = decreased expression
• yellow = no change 
• no colour = no expression 

colours are not always the same

Question 22

how do activators work?

Answer 22

promote binding of an additional activator

stimulate complex assembly - recruitment

release stalled RNA polymerase - stimulate activity

modulation of chromatin

Question 23

what is a mediator?

Answer 23

activation requires additional factors - mediator complex

Question 24

structure of mediators?

Answer 24

very large complex of 22 polypeptides

can exist on its own or associated with RNA pol II - through CTD

composed of 3 domains - head, middle and tail

Question 25

how do mediators work?

Answer 25

many activators interact with specific mediator subunits mediator provides a bridge between activators and RNA pol II • aids recruitment of RNA pol II and therefore enhance PIC formation