RR4: Proteins required to initiate eukaryotic transciption

Question 1

what is a run off assay? (single sentence definition)

Answer 1

DNA template driven by a well known characterized strong promoter

Question 2

how does a run off assay work?

Answer 2

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

Question 3

how can the level of product by run off assay be quantified/assessed and what does it say about transcription?

Answer 3

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

Question 4

how can the transcription factors be separated after a run off assay?

Answer 4

by liquid chromatography

Question 5

what are the general transcription factors? (minimal set)

Answer 5

TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH
PolII

Question 6

which is the easiest transcription factor to purify and what does it interact with?

Answer 6

TFIID is the easiest to purify
interacts with the TATA box

Question 7

which subunit of the TFIID multimeric protein is responsible for the binding to the TATA box?

Answer 7

TBP (tatabox binding protein)

Question 8

where is TBP situated in TFIID?

Answer 8

at the core

Question 9

what can TBP do in vitro?

Answer 9

alone, it is sufficient to bring in the other transcription factors and carry out transcription in vitro

Question 10

what is needed for in vivo transcription?

Answer 10

TFIID is needed for activated in vivo transcription

Question 11

what is the shape of TBP and how does it interact with DNA?

Answer 11

shaped like a saddle
binds to the minor groove and distorts the double helix, causes a kink

Question 12

what could that kink created by TBP contribute to?

Answer 12

may contribute to the recognition of that region by other transcription factors

Question 13

what classes of RNA is TBP/TFIID involved in?

Answer 13

involved in all classes
Class 1: rRNA (pol1)
Class 2: tRNA (pol3)

Question 14

what is the rest of TFIID made of? and what do some of those subunits do?

Answer 14

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)

Question 15

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)

Answer 15

1. TFIID recognises the TATA box
2. in combination with TFIIB and TFIIA, it forms a very stable structure around the TATA box
3. TFIIB interacts with the DNA, TFIIA stabilizes the whole thing
4. DNA is dramatically changed, structural kink around where those TFII proteins are associated
5. RNA polymerase enters (may always be associated with TFIIF, they have strong affinity for each other
6. RNA Pol/TFIIF enters the stable complex with interaction with TFIIB
7. now it is called the core pre-initiation complex, which is very stable
8. TFIIE recognises that complex and enters, followed by TFIIH
9. once TFIIH enters, it is called the pre-initiation complex

Question 16

how can the pre-initiation complex go from closed to open, and what does the open complex signify?

Answer 16

if closed complex is provided with adenosine containing energy source (ATP or dATP) and TFIIH
the open complex corresponds to the melting/opening of the DNA so the template strand can be accessed to by polymerase II
generates the transcription bubble
if rNTPs given, PolII will take off, initiation to elongation

Question 17

what happens to the complex after elongation starts?

Answer 17

the CTD of Polymerase II becomes heavily phosphorylated (coincident with inititiation)
many of the TFs leave the promoter, but TFIID sticks around
another series of factors join RNA polymerase II to enhance elongation
RNA PolII goes into an elongating mode

Question 18

what is present within TFIIH that allows access to the DNA template strand?

Answer 18

TFIIH has two helicases
one of them, XPB is responsible for acting to open up the DNA to allow RNA polymerase II to access the template strand

Question 19

what does TFIIH contain?

Answer 19

XPB and XPD, two large helicases
p62, p52, p44
cdk7
cyclin H
p34
MAT1

Question 20

what are XPB and XPD required for?

Answer 20

the repair of thymine dimers

Question 21

what is p44 associated with?

Answer 21

DNA repair

Question 22

what is the subunit of TFIIH that was found later and what is it responsible for?

Answer 22

TTDA-p8
responsible for the disease that causes brittle hair brittle nails and DNA repair defect

Question 23

what are the different functions of TFIIH?

Answer 23

essential transcription factor
essential DNA repair factor
cell cycle

Question 24

which subunits of TFIIH are involved in the cell cycle and what do they do?

Answer 24

cdk7
cyclin H
Mat1
form a triad that is absolutely required for the activation by phosphorylation of two or maybe more very important cell cycle cyclin dependent kinases
needed to drive the cell cycle forward

Question 25

what are the two helicases in TFIIH involved in?

Answer 25

xeroderma pigmentosum
patients are unable to repair thymine dimers (deffective nucleotide excision repair mechanism)
have defects
sensitive to UV

Question 26

what is the special feature that only TFIIH has compared to other TFs?

Answer 26

it has ATP dependent enzymatic activities

Question 27

why are certain regions of the genome repaired faster than others?

Answer 27

effectiveness of repair is associated with heavily transcribed regions
since TFIIH is there, it can repair those regions
TFIIH contributes to transcription coupled DNA repair

Question 28

where does TFIIH go when polymerase II elongates?

Answer 28

it does not follow it

Question 29

what is TFIIH also responsible for?

Answer 29

the phosphorylation of CTD