CMB2001/L02 Activators Flashcards
What 3 elements promote efficiency of transcription?
Core promoter element
Initiator
Activator binding sites
What is the function of UAS/enhancer elements?
Activate transcription (from basal level) for higher efficiency
What is the role of a factor in a common sequence element?
Aids binding of common sequence elements to transcription factors/activators
What is the sequence and factor associated with the GC box?
GGGCGG
Sp1
What is the sequence and factor associated with the octamer?
ATTTGCAT
Oct-1
What is the sequence and factor associated with the CAAT box?
GGCCAATCT
NFY
Where are common sequence elements often located?
Close to core promoter
When do common sequence elements become active?
They are constitutively active (continually)
Name 2 response elements.
SRE
HSE
What induces and binds SRE?
Induced by growth factors
Binds serum response factor (SRF)
What induces and binds HSE?
Induced by heat shock
Binds heat shock factor
Describe combinatorial control of transcription.
The type and combination of elements dictates when and at what level a gene is transcribed
What location and orientation do enhancers work in?
All locations/orientations
How are activators associated with enhancers/UAS enhancers brought into contact with GTF/DNA poll II at the core promoter?
By looping out of the intervening DNA
How many DNA binding domains and activation domains are on a strand of DNA?
1 DNA binding domain
Maybe more than 1 activation domain
Give 2 DNA binding domains.
Leucine zipper
Homeodomain
Helix loop helix
Zinc finger
How are activation domains often characterised?
According to amino acid composition
Give 3 features of activation domains.
Lack of sequence conservation and structural information
Generally thought to be unstructured
Contain multiple short segments that work together in additive fashion
Interact with other proteins in transcriptional machinery
How do activation domains promote transcription? (2)
Recruit transcription factors and coactivators
Modify chromatin structure to make DNA more accessible for transcription
How are activators analysed in vitro by gel shift/electrophoretic mobility shift assays?
Activator and radiolabelled probe DNA run on non-denaturing acrylamide gel
Gel separates based on size and mass
Only measure ability to bind DNA, not transcription
How are activators analysed in vitro by transcription assays? (2)
RNA pol II, GTFs, DNA template, radiolabelled rNTPs assayed
Requires activator to have functional DNA binding domain and activation domain
How are activators analysed in vivo? (3)
Reporter assays
Plasmid 1 encodes protein X
Reporter gene of plasmid 2 activated by protein X binding
Reporter gene transcripts produced
Describe the 5 steps of chromatin immunoprecipitation.
Crosslink bound proteins to DNA
Isolate chromatin and shear DNA
Precipitate chromatin with protein-specific antibody
Reverse cross-link and digest protein
Analyse DNA using PCR or sequencing
Give 4 ways in which activators work.
Cooperative binding- promote binding of additional activator (uncommon)
Stimulate complex assembly (major)
Release stalled RNA pol (stimulate activity)
Modulation of chromatin
Which components of the PIC complex do activators interact with to promote assembly? (3)
TFIID (via TAFs)
TFIIB
Mediator
What is the mediator complex?
An additional factor required for transcription activation in minimal in vitro transcription systems
Describe the structure of mediator. (3)
22 polypeptides
Can exist on its own or associated with RNA pol II through C-terminal domain
Composed of 3 domains - head, middle, tail
Describe the function of mediator. (2)
Provides a bridge between activators and RNA pol II
Mediator-activator interactions aid recruitment of RNA pol II to enhance PIC formation
Where can RNA pol II stall?
On or near the promoter
Give an example of an activator releasing stalled RNA pol. (3)
Heat shock genes e.g., hsp70
IN absence of heat shock, RNA pol II pauses after -50nts
Heat shock activates HSF transcription factor which interacts with RNA pol II
Releases it from the pause
