lecture 2 Flashcards
what are the two classes of UAS enhancer elements?
common sequence elements & response elements
what are common sequence elements?
-located close to core promoter
-bind activators that are relatively abundant in cell and continually active
-GC box, octamer, CAAT box
what are response elements?
-bind factors whose activity is controlled response to specific stimuli
SRE = binds inducer, growth factors
HSE = binds inducer, heat shock
what does the type and combination of elements dictate in transcription?
when and at what level the gene is transcribed
what happens in an absence of heat shock?
low levels of transcription
how does the enhancer location work?
-independently of its position
-if enhancer binds upstream it still activates transcription irrespective of position
how do activators contact the basal transcription machinery?
the activator binds to enhancer and tracks along DNA and contacts RNA polymerase and GTFs located at basal promoter
what’s the looping method of contacting the basal transcription machinery?
-bring activator at UAS into close contact with basal promoter by looping the DNA and transcription machinery
what are the components of eukaryotic activators?
single binding domain, activation domain (can have more than one), flexible protein domain to link the two
what are the properties of activation domains?
-lack of sequence conservation and structural information
-unstructured until interacts with protein
-multiple short segments that work in additive fashion
how are electrophoretic mobility shift assays used to analyse activators?
-protein is isolated to find out what it binds to
-purified activator is mixed with probe DNA
-lower mobility = runs quickly through gel = free DNA
-if protein is bound= larger = runs through gel much slower
how are transcription assays used to analyse activators?
-purified polymerase GTFs and DNA template have radio labelled nucleotides added
-absence of activator = no radio labelled transcript produced
what is required to stimulate transcription of RNA transcript?
-functional DNA binding domain
-functional activation domain
how are reporter assays used to analyse activators (in vivo approach)?
-one plasmid expressing protein x (activator) and another has reporter gene under control of presumed binding site
-introduced into cell and transcription factor expressed
-if binds, transcription of reporter gene is activated and gets reporter gene activity
what does chromatin immunoprecipitation (ChIP) show ?
where proteins bind to dna within a cell
what occurs in chromatin immunoprecipitation (ChIP)?
-cross linking agent added to cells to crosslink bound proteins to DNA
-cells broken up and DNA sheared
-antibody specific to the protein of interest used to precipitate activated protein that’s bound to binding site
-left with the DNA binding sites for the protein only
how do activators work synergistically?
promote binding of an additional activator
how do activators work (1)?
- stimulate complex assembly of PIC (recruitment)
- promotes formation of transcription complex at the basal receptor
-promoter will increase efficiency and production of RNA
what is mediator?
-the middle part that helps activators activate transcription in minimal conditions
what is the composition of the mediator?
-large complex of 22 polypeptides
-can exist on own or associated with RNA pol II through C-terminal domain
-composed of 3 domains: head, middle, tail
what is the function of the mediator?
-provides BRIDGE between activators and RNA pol II
-aids RNA polymerase recruitment
-enhances PIC formation so assembly of PIC so imitates transcription
how do activators work (3)?
3.release stalled RNA polymerase to stimulate activity
-RNA pol can stall near promoter (common in heat shock genes)
-heat shock interacts with stalled polymerase and releases to allow it to elongate
how do activators work(4)?
- modulation of chromatin
-has big impact on whether it occurs
-brings about remodelling of chromatin to allow complex formation
