Building the preinitiation complex

1
Eukaryotic Transcription Part 4 CTD of
RNAPII role of TFIIF, TFIIE, TFIIH a structural
Model for the PIC role of the CTD of RNAPII in
initiation transcription cycle Holoenzyme vs.
multistep assembly
2
Note that RAP30 shows similarity to bacterial s
factor domains 2 and 3.
Model for PIC structure
Mapping the Location of TFIIB within the RNA
Polymerase II Transcription Preinitiation Complex
A Model for the Structure of the PIC, (2004)
Chen, H.-T. Hahn, S., Cell 119169-180.
3
CTD of RNA pol II large subunit
1. The carboxy-terminal domain (CTD) of the
largest subunit (? homolog) consists of an array
of amino acid repeats (7 aa each) of the sequence
Tyr-Ser-Pro-Thr-Ser-Pro-Ser. 52 repeats in
mammals, 42 in plants, 26 in yeast. 2. It must
be unphosphorylated for RNA pol II to join the
PIC. Becomes hyperphosphorylated by TFIIH. This
process is linked to the transition between
initiation and elongation. 3. Binds mediator
complex.
pol II
CTD
4
Basal factors
TFIIF (RAP30 RAP74 tetramer) 1. promoter
targeting of RNA pol II by binding to TFIIB, and
reduces nonspecific DNA interactions of pol
II. 2. involved in initiation and elongation
decreases pausing and protects elongation complex
from arrest.
CTD
RNA pol II
TFIIF
-N
Zn-ribbon
5
Basal factors
TFIIF (RAP30 RAP74) 3. stimulates phosphatase
activity which removes phosphates from the CTD.
Stimulation of phosphatase only occurs in the
absence of binding to IIB (after initiation).
elongation
CTD
RNA pol II
-
-
TFIIF
5
RNA
6
TFIIE (34 57 kDa subunits tetramer) 1. binds
RNA pol II and recruits TIIH. 2. modulates TFIIH
helicase, ATPase and kinase activities. 3.
Assists TFIIH in promoter melting.
Basal factors
CTD
RNA pol II
TFIIF
TFIIE
TFIIH
7
Basal factors
TFIIH (9 subunits) 1. closely associated with
TFIIE binding and regulation. 2. promoter
melting through helicase activity (3-5). 3.
promoter clearance by CTD kinase activity. 4.
functions in nucleotide excision repair (NER
5-3 helicase). 5. mutations can result in xerma
pigmentosa in humans.
RNA pol II
CTD
TFIIE
TFIIH
8
Basal factors
TFIIH (9 subunits) 1. TFIIH core 2 helicases
(3 to 5 and 5 to 3) and DNA binding activity
6-7 subunits. 2. protein kinase complex called
TFIIK in yeast or CAK 3 subunits.
Phosphorylates the CTD of RNA pol II large
subunit, and activates cyclin-dependent kinases
(CDKs). CAK (cdk activating kinase)
Helicases require ATP to melt the DNA.
core

cdk7
CAK module
Mat1
cycH

• CAK (3 subunits)
• CTD kinase (cdk7)
• Cyclin H
• Mat1

Note most of the CAK in the cell is not
associated with TFIIH.
9
TFIIH involvement in nucleotide excision repair
of DNA cell cycle
phosphorylates CTD
CAK (cdk7, cycH, Mat1)
CAK activity (CDK activating kinase) activates
cyclin-dependent kinases (CDKs)
core IIH (helicases)
Note only the core is involved in both
transcription and NER.
transduces signal to cell-cycle
machinery/apoptosis
preferential repair of template strand
10
Basal factors
TFIIE/H functions in transcription 1.
phosphorylates CTD which facilitates promoter
clearance.
CTD
-
-
-
-
-
TFIIE
3-5 helicase (transcription) 5-3 helicase
(NER).
TFIIH
-9
1
11
Basal factors
TFIIE/H functions in transcription 3. required
for formation of first phosphodiester bond in RNA
synthesis.
CTD
-
-
-
-
-
TFIIE
TFIIH
5
-9
8
pU-pA-
12
Human TFIIH EM-derived structure
CAK
helicase
helicase
NER
Molecular Structure of Human TFIIH (2002) Patrick
Schultz, Se bastien Fribourg, Arnaud Poterszman,
Ve ronique Mallouh, Moras, and Jean Marc Egly,
Cell 120 599-607.
13
Two Models for the Activation of Transcription
1. Multistep Assembly
Activator
Activator
14
Two Models for the Activation of Transcription
1. Multistep Assembly
E-H
pol II
Activator
F
15
Two Models for the Activation of Transcription
1. Multistep Assembly
Activator
pol II
F
E-H
16
Two Models for the Activation of Transcription
2. Holoenzyme
holoenzyme
Activator
IID recruited separately
17
Two Models for the Activation of Transcription
2. Holoenzyme
mediator
Activator
pol II
F
E-H
Note the holoenzyme model is largely discredited
now due to the low abundance of holoenzyme in
cells and the results of ChIP assays that show
multi-step recruitment events.
18
RNA pol II exists in a complex with Mediator and
TFIIF
mediator
mediator
RNA pol II
mediator
(12 subunits)
CTD
RAP30
TFIIF
Hypophosphorylated CTD
RAP74
Hyperphosphorylated CTD
Mediator is a complex of 20-30 proteins that are
involved in facilitating active and basal
transcription. Mediator only attaches to the
hypophosphorylated CTD (under phosphorylated).
19
Transcription Cycle
step 1
Mediator
RNA pol II/mediator joins the PIC if the CTD is
hypophosphorylated.
RNA pol II
CTD
(under phosphorylated)
IIF
20
Transcription Cycle
step 2
Mediator
TFIIE TFIIH join the complex
RNA pol II
CTD
TFIIE
TFIIH
21
Building the preinitiation complex (PIC)
RNA pol II with meidator plus TFIIF join the
preinitiation complex. TFIIB binds to
TFIIF. TFIIF also makes contacts with TAF1, TAF5,
and TAF6
mediator
RNA pol II
(12 subunits)
CTD
RAP30
TFIIF
RAP74
TBP
TFIIA
TFIIB
22
Role of TFIIB and TFIIF in binding RNA pol II
1. TFIIF attaches to N-terminus of TFIIB (Zn
finger). 2. RNA pol II attaches to TFIIB core
(R1/R2). 3. CTD binds TBP
RNA pol II
CTD
TFIIF
TFIIB
TBP
TFIIA
Note mediator not shown.
23
Building the preinitiation complex (PIC)
TFIIE and TFIIH are last to join the PIC. TFIIE
attaches TFIIH to RNA polymerase and modulates
the activity of H. TFIIH has 9 subunits including
2 helicases and a kinase (cdk7) that
posphorylates the CTD of pol II.
TFIIH
TFIIE
RNA pol II
TFIIF
TBP
TFIIA
TFIIB
Note CTD binds TBP mediator not shown.
24
Three attachments of RNA pol II to the
preinitiation complex 1. TFIIF binds TFIIB
n-terminus (Zn finger) 2. Carboxy terminal domain
of large subunit of RNA pol II (CTD) binds
TBP. 3. direct interaction with TFIIB.
RNA pol II
CTD
TBP
TFIIF
TFIIA
TFIIE
R2
TFIIH
R1
TFIIB
Note not shown are mediator and TAF
interactions.
25
Transcription Cycle
Mediator
RNA pol II
CTD kinase complex (TFIIH) phosphorylates the CTD
-
-
-
-
TFIIE
TFIIH
Note CTD phosphorylation acts as the trigger
that ends the initiation phase and begins
elongation.
26
Transcription Cycle
Mediator looses affinity for the CTD after the
CTD is hyperphosphorylated.
step 4
RNA pol II
Mediator
Hyperphos. CTD dislodges from TBP and from
Mediator.
TFIIE
TFIIH
27
Transcription Cycle
RNA pol II initiates and clears the promoter with
CTD hyperphosphorylated
Note TFIIE, TFIIH, TFIIA, TFIID and mediator
remain at the promoter.
Med.
TFIIE
TFIIH
RNA pol II
-
-
-
-
-
-
TFIIF
TFIIB
5
28
Transcription Cycle
The CTD is dephosphorylated by a phosphatase
activity stimulated by TFIIF (in the absence of
TFIIB).
-
-
-
-
RNA pol II
CTD
TFIIF
29
Transcription Cycle
Mediator
Termination RNA pol II/TFIIF complex leaves the
template, forms complex with the mediator, and
reinitiates with the CTD in the
underphosphorylated state.
RNA pol II
CTD
TFIIF
30
Reinitiation Scaffold complex
What 3 components leave?
RNA pol II
Mediator
TFIIF
TFIIE
TFIID
TFIIB
TFIIH
TBP
TATAA
Activator
TFIIA
Yudkovsky, Ranish, Hahn (2000) Nature 408
225-229.
31
Yeast nuclear extract Gal4-AH
Template immobilized on magnetic beads
Fig. 1 Scaffold contains TFIID, TFIIA, Mediator,
TFIIH and TFIIE. 40 min prebinding followed by
wash. Add nucleotides for 2 min. (allows only one
round of transcription). Scaffold proteins
remaining at the promoter
Note that adding just ATP also caused
disassociation of the PIC.
Yudkovsky, Ranish, Hahn (2000) Nature 408
225-229.
32
References used for the Eucarya lectures
1) Luders et al. (2000) The ubiquitin-related
BAG-1 provides a link between the molecular
chaperones Hsc70/Hsp70 and the proteasome, J.
Biol. Chem. 275 4613-4617. 2) Jentsch
Pyrowolakis (2000) Ubiquitin and its kin how
close are the family ties? Trends Cell Biol. 10
335-342. 3) Vierstra (1996) Proteolysis
mechanisms and functions, Plant Mol. Biol. 32
275-302. 4) Russell, Reed, Huang, Friedberg,
Johnston (1999) The 19S regulatory complex of the
proteasome functions independently of proteolysis
in nucleotide excision repair, Mol. Cell 3
687-695. 5) Guzder, Bailly, Sung, Prakash,
Prakash (1995) Yeast DNA repair protein RAD23
promotes complex formation between transcription
factor TFIIH and DNA damage recognition factor
RAD14, J. Biol. Chem. 270 8385-8388. 6) Kuras,
Kosa, Mencia, Struhl (2000) TAF-containing and
TAF-independent forms of transcriptionally active
TBP in vivo, Science 288 1244-1248. 7) Li,
Bhaumik, Green (2000) Distinct classes of yeast
promoters revealed by differential TAF
recruitment, Science 288 1242-1244.