activators And Txn Activation

Question 1

What are the 2 classes of UAS/enhancers

Answer 1

Common sequence elements

Response elements

Question 2

Give 3 examples of common sequence elements and what factors/activators they bind to

Answer 2

Gc box - sp1 binds to it

Octamer - Oct 1

Caat box - nfy

Question 3

Are common sequence elements like gc box proximal or distal from core promoter

Answer 3

Proximal

Question 4

Are activators like sp1 which bind to common sequence elements constantly active and unchanged by stimuli

Answer 4

Yes

They don’t help promoter respond to a specific stimuli

Question 5

What are response elements

Answer 5

Distal UAS which help promoter/gene exp respond to specific stimuli eg heat shock

Question 6

Give 2 examples of response elements and their inducers and what they bind to

Answer 6

Serum response factor SRF
Binds to serum response elements SRE
When it is induced by growth factors

Heat shock factor HSF
Binds to heat shock element HSE
When heat shock occurs

Turns on genes for heat shock

Question 7

Can response elements like hse combine with common sequence elements like gc or octamer

Answer 7

Yes. Common are always proximal tho

Question 8

What happens to common sequence elements like gc or oct or caat when heat shock occurs

Answer 8

No change. Txn stays at a level

Question 9

Does location of UAS or the orientation affect ability to activate promoter

Answer 9

No

They can even be downstream from the start site

Question 10

What are the 3 components of activators like sp1

Answer 10

Flexible protein domain (long string)

Activation domain (1+)

Dna binding domain (just 1)

Question 11

Why is activation domain unstructured and not conserved

Answer 11

Dependant on AA composition

Question 12

Name 3 types of activation domains

Answer 12

Acidic patch (asp or glu - charge)
Glutamine rich
Proline rich

Question 13

What activator has an acidic patch activating domain

Answer 13

Vp16

Question 14

What activation domain does sp1 have

Answer 14

Glutamine rich

Question 15

Which part of activators interacts with proteins like TAFs

Answer 15

Activation domain

Question 16

Give 4 types of dna binding domains on activators

Answer 16

Zinc finger
Leucine zip
Helix loop helix
Homeodomain

Question 17

What are the 3 in vitro ways to analyse activators

Answer 17

Dna footprinting
Electrophoretic mobility shift assay (gel shift)
Transcription assay

Question 18

How does dna footprinting show there’s an activator bound to dna

Answer 18

Because DNase can’t cut the dna and so there in a section in the gel where bands don’t appear

Question 19

How does electrophoretic mobility shift assays identify if activator is present

Answer 19

The dna is run on gel and if protein is bound this complex runs slower on the gel

Question 20

Electrophoretic mobility shift assays and dna footprinting test ability to bind but what doesn’t it test

Answer 20

Ability to activate

Question 21

How do transcription assays work to test ability of activation

Answer 21

Put all things needed for txn Eg gtf pol II and rntps rsdiolabelled

Add an activator

Rna product size picked up using radiography

Question 22

How are in vivo reporter assays used to work out activator ability

Answer 22

Plasmid 1 which has gene for the activator x and plasmid 2 which a reporter gene Eg gfp is contransected into a cell

The gene activator x is transcribed and translated

The ability of this gene to then activate txn and translation of the reporter gene from plasmid 2 is detected post translation

Question 23

Other than reporter analysis, what other in vivo is used to analyse activator

Answer 23

Chip chromatin immunoprecipitation

Question 24

What does chip do

Answer 24

Identifies where on dna and what gene activators bind to

Identifies all binding sites

Question 25

What are the steps of chip

Answer 25

Cross link the proteins to the dna (glue them)

Shear the dna via sonic stuop

Immunoprecipitate with protein specific antibody

Isolate the antibody protein dna complex

Reverse cross link via heat to seperate purified protein and dna

Question 26

When dna and protein are reverse cross linked what happens with the dna

Answer 26

Goes for sequencing to find the dna bs

Question 27

What are the 4 ways activators work

Answer 27

1- cooperative binding
2- pic assembly stimulation
3- post recruitment activation (stalling)
4- chromatin remodelling

Question 28

Explain cooperative binding

Answer 28

1 activator can help another bind

Question 29

Which 3 things do activators interact with to stimulate pic assembly

Answer 29

II d (via TAFs and activation domain)
II b
Mediator complex

Question 30

What is the mediator complex

Answer 30

22 polypeptide complex which connects the activator to the pic

Question 31

What does the mediator attach to on rna pol

Answer 31

CTD in rpb1

Question 32

What are the 3 domains of mediator complex

Answer 32

Head middle tail (interact with diff activators)

Question 33

What is post recruitment activation and give example

Answer 33

Some genes Eg heat shock genes the rna pol will stall after promoter clearance if there’s no heat shock

Activators can help release it

When heat shock occurs this can promote activators like hsf to the hse and stop stalling via interaction with rna pol via the CTD