lecture 2 Flashcards
basal transcription alone results in
low/inactive transcription
upstream activator sequences (uas) / enhancer elements effects
act as binding site of activator proteins which simulates transcription initiation
what are the common sequence UAS/ enhancer elements
GC box, Octamer and CAAT box
what transcription factor does GC box bind to
Sp1
what factor does octamer bind to
Oct-1
what factor does CAAT box bind to
NFY
where are enhancer elements located
close to the core promoter (promoter proximal)
response elements are
in the enhancer region that are able to bind to transcription factors and regulate transcription of genes
response element SRE binds to ?
serum response factor SRF and induced when growth factors are present
response element HSE binds to ?
heat shock factor, induced by heat shock
response elements bind
factors whose activity is controlled in response to specific stimuli
enhancer can be located?
upstream or downstream of the gene they control and at large distances
activator protein is the same as
transcription factors
activators need 2 domains..
DNA binding domain and activation domain
in prokaryotic activators the binding domain and activation domain are
integrated into one region
in eukaryotic activators they are
separate and they are linked by a flexible protein domain
generally how many binding and activation domains are there in eukaryotic activators
one binding domain and multiple activation domains
examples on dna binding domains are
leucine zipper, zinc finger, homeodomain, helix loop helix
activation domains are characterised according to their
amino acid sequence
activation domains interact with
other proteins in the transcriptional machinery eg TAFs
electrophoretic mobility shift assays are used to measure the
ability for an activator to bind to DNA
radiolabeled probe DNA and activator is run on
non denaturing acrylamide gel
you get two bands if the activator has bound to the DNA and it will of moved down the gel more or less?
less as its a bigger complex
transcription assays show
ability for activator to initiate transcription
transcription assays require
RNA pol II, GTFs (general transcription factors(TFIIX)), DNA template, radiolabeled rNTPs
if the activator works you will see
an radiolabeled RNA transcript in the acrylamide gel
in vivo method
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
chromatin immunoprecipitation (ChIP) in vivo method allows us to understand
where transcription factors bind in cells
add cross linking agent which
cross links activator protein to DNA
add cross linking agent which
cross links activator protein to particular DNA site
next you break down the cell and shear DNA into little chunks and then
purify blue sequences by using a specific antibody to the activator
reverse cross links by heating
leaving enriched sample of all places n the dna where the activator binds
sequence the data using
ChIP-Seq to identify all the binding sites for an activator
how do activators work 1. binding of first activator to UAS promotes
binding of a second activator to a different UAS
how do activators work 2. binding stimulate recruitment of RNA polymerase, what components of PIC complex so activators interact with
TFIID (via TAFs), TFIIB, mediator (major target)
what is mediator
mediator is a large complex of 22 polypeptides, can exist on its own or associated with RNA pol II
how many domains to mediator have
3, head, middle and tail
mediator subunits interact with
many activators
mediator provides a bridge between
activators and RNA pol II
mediator - activator interactions aid recruitment of
RNA pol II and therefore enhance PIC formation stimulating transcription
the polymerase can stall near to the promoter, this is released by
active activator proteins release stalled RNA pol II
heat shock factor binds to activator (HSF transcription factor) and it interacts with RNA pol II
and releases it from the pause
