activators And Txn Activation Flashcards
What are the 2 classes of UAS/enhancers
Common sequence elements
Response elements
Give 3 examples of common sequence elements and what factors/activators they bind to
Gc box - sp1 binds to it
Octamer - Oct 1
Caat box - nfy
Are common sequence elements like gc box proximal or distal from core promoter
Proximal
Are activators like sp1 which bind to common sequence elements constantly active and unchanged by stimuli
Yes
They don’t help promoter respond to a specific stimuli
What are response elements
Distal UAS which help promoter/gene exp respond to specific stimuli eg heat shock
Give 2 examples of response elements and their inducers and what they bind to
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
Can response elements like hse combine with common sequence elements like gc or octamer
Yes. Common are always proximal tho
What happens to common sequence elements like gc or oct or caat when heat shock occurs
No change. Txn stays at a level
Does location of UAS or the orientation affect ability to activate promoter
No
They can even be downstream from the start site
What are the 3 components of activators like sp1
Flexible protein domain (long string)
Activation domain (1+)
Dna binding domain (just 1)
Why is activation domain unstructured and not conserved
Dependant on AA composition
Name 3 types of activation domains
Acidic patch (asp or glu - charge)
Glutamine rich
Proline rich
What activator has an acidic patch activating domain
Vp16
What activation domain does sp1 have
Glutamine rich
Which part of activators interacts with proteins like TAFs
Activation domain
Give 4 types of dna binding domains on activators
Zinc finger
Leucine zip
Helix loop helix
Homeodomain
What are the 3 in vitro ways to analyse activators
Dna footprinting
Electrophoretic mobility shift assay (gel shift)
Transcription assay
How does dna footprinting show there’s an activator bound to dna
Because DNase can’t cut the dna and so there in a section in the gel where bands don’t appear
How does electrophoretic mobility shift assays identify if activator is present
The dna is run on gel and if protein is bound this complex runs slower on the gel
Electrophoretic mobility shift assays and dna footprinting test ability to bind but what doesn’t it test
Ability to activate
How do transcription assays work to test ability of activation
Put all things needed for txn Eg gtf pol II and rntps rsdiolabelled
Add an activator
Rna product size picked up using radiography
How are in vivo reporter assays used to work out activator ability
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
Other than reporter analysis, what other in vivo is used to analyse activator
Chip chromatin immunoprecipitation
What does chip do
Identifies where on dna and what gene activators bind to
Identifies all binding sites
What are the steps of chip
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
When dna and protein are reverse cross linked what happens with the dna
Goes for sequencing to find the dna bs
What are the 4 ways activators work
1- cooperative binding
2- pic assembly stimulation
3- post recruitment activation (stalling)
4- chromatin remodelling
Explain cooperative binding
1 activator can help another bind
Which 3 things do activators interact with to stimulate pic assembly
II d (via TAFs and activation domain)
II b
Mediator complex
What is the mediator complex
22 polypeptide complex which connects the activator to the pic
What does the mediator attach to on rna pol
CTD in rpb1
What are the 3 domains of mediator complex
Head middle tail (interact with diff activators)
What is post recruitment activation and give example
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
