RR4: Proteins required to initiate eukaryotic transciption Flashcards
what is a run off assay? (single sentence definition)
DNA template driven by a well known characterized strong promoter
how does a run off assay work?
cut a plasmid with restriction enzymes at a specific site, you know how long it is
make it linear
put a well known, strong, well characterized promoter (ADMLP) upstream of that segment
all the required transcription factors will come on the promoter and transcribe that gene
the RNA polymerase drops off at the end of the section and comes back
how can the level of product by run off assay be quantified/assessed and what does it say about transcription?
more RNA made = more efficient
quantify how much RNA was made with probes (acrylamide gel electrophoresis separates them) or by adding radioactive ribonucleotides in the test tube and then doing autoradiography
how can the transcription factors be separated after a run off assay?
by liquid chromatography
what are the general transcription factors? (minimal set)
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH
PolII
which is the easiest transcription factor to purify and what does it interact with?
TFIID is the easiest to purify
interacts with the TATA box
which subunit of the TFIID multimeric protein is responsible for the binding to the TATA box?
TBP (tatabox binding protein)
where is TBP situated in TFIID?
at the core
what can TBP do in vitro?
alone, it is sufficient to bring in the other transcription factors and carry out transcription in vitro
what is needed for in vivo transcription?
TFIID is needed for activated in vivo transcription
what is the shape of TBP and how does it interact with DNA?
shaped like a saddle
binds to the minor groove and distorts the double helix, causes a kink
what could that kink created by TBP contribute to?
may contribute to the recognition of that region by other transcription factors
what classes of RNA is TBP/TFIID involved in?
involved in all classes
Class 1: rRNA (pol1)
Class 2: tRNA (pol3)
what is the rest of TFIID made of? and what do some of those subunits do?
TAFs
TBP associated factors
some of the TAFs also interact with other cis acting elements such as the initiator and the downstream promoter element
–> why it is necessary for in vivo (activated transcription)
how do all the transcription factors come together to start transcription (set up of the pre-initiation complex) (stepwise manner only applicable to in vitro)
- TFIID recognises the TATA box
- in combination with TFIIB and TFIIA, it forms a very stable structure around the TATA box
- TFIIB interacts with the DNA, TFIIA stabilizes the whole thing
- DNA is dramatically changed, structural kink around where those TFII proteins are associated
- RNA polymerase enters (may always be associated with TFIIF, they have strong affinity for each other
- RNA Pol/TFIIF enters the stable complex with interaction with TFIIB
- now it is called the core pre-initiation complex, which is very stable
- TFIIE recognises that complex and enters, followed by TFIIH
- once TFIIH enters, it is called the pre-initiation complex