17 Flashcards
What about promoters lacking TATA… how does transcription initiate
TATA binding protein on its own cant bind without a tata box so how does tfiid bind
When there is no tata box…
can either bind to :
1)INitiator
the tatabinding associated proteins are what allow tfIID to bind now to INITIATOR element and TBP positioned ina. way that it would if tata was there
-ie TAFII-250 (taf)
2) CG box:
-TAF binds to a protein which binds to the GC and interacts with other tafs to allow TBP to be anchored (since the tafs are) and TFIID is now bounded
Do most rna pol have tata box
No… sadly
TF (Transcription factor) ACTIVITY ASSAY ON EXAM!!!!!!!
Yeast Two Hybrid System
Purified transcription factor:
-determine partial AA sequence
-screen library or search data bases
-gene (cDNA) for X)
Suppose we want to check if a protein acts as a transcription factor
Happens in yeast (simplicest euk to use for testing)
We think X might be involved in transcription so what we do is Contruct two plasmids
1) With gene encoding transcription factorX (gene for X, so making X)
2) with reporter gene and X binding site
If X does act as transcription factor, X will bind (comesfrom first plasmid) and get transcriptio of the reporter gene of the other plasmid
So say that X does bind and cause transcription of reporter… once this happens we can mutate X to see what amino acids and the domain of the binding site
Proteins that affect transcription
Enhancers: activators bind here to increase transcription
-positively increase transcription
-site for binding
-work backwards or forwards cause it can loop around
Other proteins that affect it:
Co-activators and mediators: Euchromatin opens dna heterochromatin closes, by regulating this ratio we can regular it
-bind to both activators and basal apparatus (bridge)
Remember in prok vs euk
RNAP needs sigma to bind : pro (helps it recognize the promoter
Activators and repressors exist in prok (make the bidning of the pol to promoter better or worse)
Euk: rnap needs General transcription factors to bind (TFIID, etc)
activators and repessors are Transcription factors in euk
Mediators
Remmeber: every cell type has its own set of activators that activate its own set of genes, and each cell has so many enhancers too so how does rna pol recognize all of these
This is where mediator comes in: a mediator is like an adaptor…say the shape of the activator for gene A is a square and gene B is a circle, the adaptor binds to the activator and then this is what binds to the rna pol
so if acell wants to use a certain activator, it would make the mediator for it
-able to bind any rna pol on one side but only specific activators on the other side
Complex: complex necessary for the assembly of the pre initiation complex (rnap2 and gtfs)
how can stem cells have the ability to be anything
they have a complete set of mediators so that they can turn on any gene in the genome
Transcriptionfactors (not general)
All need to :
1. bind to dna
2. Be able to activate or repress transcription
Always have two Domains
1. DNA- Binding domain (DBD):One that binds to dna
2. Transcription Activation DOmain (AD): one that binds to rna pol;interact wiht other proteins to stimulate transcription from a nearby promoter
Some also have:
Dimerization domans:
Allow for great diversity and complexity of factors
Ligan Binding DOmains: binding of accessory smll molecule regulates transcription actiity (think lac binding causes it to unbind so the transcription factor needs place for this to bind so that it can be controlled)
What are domains and motifs
Domains: tertiary structures of proteins organized into DISTINCT regions of the protein, can be active or stable on their own when removed from rest of protein
Motifs: combination fo secondary structures that are organized into 3d strucutres inside domain (responsible for functioning)
Independence of DNA binding and Transcription activation domains
Experiments with chimeric (hybrid ) proteins: Activating domans and dna binding domain are seperate (independent of eachother) we see this with this experiment:
If u chop off activator: notranscription bc rna pol cant bind
if u change binding sites and put it in same spot : stimulates transcription, shows that the dna binding domain does not need that specific activating domainto work, just needs one in general
SOOO if : dna binding domain from repressor (ie lex A), activation domain from activator (ie Gal4), it will function as a ACTIVATOR (GAL4) even though the binding domain is from the repressor
Slide 9
sooo activation domain is important for ____ and dna binding domain is
Activation domain is important only for activation and DNA binding domain only for binding – they are independent
Dimerization domain
Combination of two different activation domains into a new regulatroy combination, allows recognitions of different binding sites
since activator binding site and dna binding site can act independently, we can make new combinations
-can use small amount of activators to act on many different sites
-also works with inhibitors, same idea
Heterodimerization of AD: Combinatorial control
ie if the site was now blue and pik, means that even a molecule that have one blue can cause it to do its effects
Ligand binding domain
Ligands: any type of effector molecules
ie CAMP
Enhancers vs upstream element6s
How are enhancers different from upstream elements that do something for transciprtion:
ie GC boxes, need to be oriented a certain wau, upstream sequences have an orientation!and need to be really close, because the proteins need to touch the rna pol
enhancers: can be any distance away and oriented any way
think: enhancers have it easy, think a really hot guy asking someone out, he doesnt have to do much
upstream sequences: have to do alot, they are like uglier guys who have to get flowers, pay for the meal etv
Enhancers
can be any distance away
-except close cuase it cant bend the dna
Enhancesome: binding creates this,cooperative bidning of dna
