GTFs and PIC
assembly
T
TB
TAT
Promote
MBV4230
GTFs and PIC assembly

General transcription factors (GTFs)

make RNAPII capable of selective initiation in vitro
TB
TFII
+


Highly conserved
RNAPII+GTFs = ca. 30 polypeptides

≈ 2 MDa
TFII
TFII
TFII
TFII
= PIC
Correct
initiation
of trx
in vitro
Odd S. Gabrielsen
MBV4230
Linear assembly of PIC the preinitiation complex





A specific order of operation:
 DAB-Fpol-EH
Nucleation

TFIID+TATA form an “initial committed complex”

TAFs + INR may also initiate PIC-assembly

Common: a core sequence is recognized by a seq.spes.GTF
Link

initial complex recognized by TFIIB

With TFIIB bound, the complex becomes accessible to RNAPII
RNAPII recruitment

Assembly of RNAPII assisted by TFIIF

Minimal initiation complex formed
Maturation to complete trx competent PIC

Minimal initiation complex (DABF-pol) NOT trx.competent

Recruitment of TFIIH and TFIIE necessary

This step is unique for RNAPII
Odd S. Gabrielsen
MBV4230
Alternatives to
linear PIC-assembly
Alternative
Nucleation events
Nucleation
Link
RNAPII recruitment
Holoenzyme
Maturation
2-step
alternative
Odd S. Gabrielsen
MBV4230
TBP [TFIID] function

Binds TATA - main sequence
recognition event during PIC assembly







Binds a variety of different TATA-like sequences
A slow binding reaction
Other factors
N
minor groove contact
binds as monomer
DNA
Affinity of TBP for TATA contributes to
promoter strength
Binds also several other polypeptides

activators (Sp1, Tax1, E1A)


TAFs (dTAF110, dTAF40)
GTFs (TFIIB, TFIIA)

inhibitors
TBP = universal TF involved in all three
RNA polymerase systems

TBP i SL1, TFIID, TFIIIB
Odd S. Gabrielsen
MBV4230
TBP versus TFIID

Subunit-structure



TAFs
TFIID = TBP + multiple TAFs
mammalian TFIID: 750 kDa (II), 300 kDa (III) and 200 kDa (I)
TBP only a small core in the TFIID complex

human 38 kDa, yeast 27 kDa, Arabidopsis 22 kDa
TBP

TBP = N-term divergent domain + C-term. conserved domain
 C-term domain 180 aa symmetric


N-term domain divergent

N
Carries all essential functions
probably involved in regulating DNA binding
TFIID
TBP
Odd S. Gabrielsen
MBV4230
TBPs saddle-structure
Convex
surface
protein
Concave
inside
DN
A Stigbøyler
stirrups
3D: saddle-structure
•
•
•
•
Twofold symmetry - form of a saddle.
Concave inside binds DNA in minor groove through a 10-stranded antiparallel β-sheet
Convex surface binds other GTFs via 4 α-helixes
loop (“stirrup”) on each side with Phe side-chains intercalating in DNA
Odd S. Gabrielsen
MBV4230
TBPs effect on DNA

DNA-structure is distorted upon TBP binding




DNA severely bended, unwinded and distorted
DNA shaped by TBP´s β-sheet
The intercalating Phe-residues contributes to kink
Effect?


Upstream and downstream elements brought closer together
incompatible with nucleosome structure
Not like
this
.. but this way
Odd S. Gabrielsen
MBV4230
A Two-Step Mechanism of TBP
Binding to DNA

First step


Full-length TBPWT first
binds to TATA box to form
an unbent TBP-TATA box
complex.
Second step


Then, this unbent complex
slowly forms the bent TBPTATA box complex.
TFIIB can directly recognize
the unbent and/or bent TBPTATA-complexes to form
the bent TBP-TATA box
complex.
Odd S. Gabrielsen
MBV4230
TFIIB

Functions in PIC-assembly as adaptor - a
molecular bridge that couples TBP-TATA with
RNAPII

TFIIB recognizes the distorted TBP-TATA complex

contacts DNA on both sides of TBP-TATA

upstream via major groove (BRE) and downstream via minor groove
Provides directionality to the complex through assymmetric binding


TFIIB mediates RNAPII binding

interaction also with TFIIF
TAT
A
BRE
TFIIB

+1
TSS
Function in initiation: “Measures” distance TATA - TSS
Odd S. Gabrielsen
MBV4230
TFIIB

TFIIB also contact point for activators


VP16, Steroid hormone receptorer, fushi tarazu, TAF40
TFIIB-BRE: a repressive interaction?

The BRE was recently reported to repress basal transcription, with
activator-mediated disruption of the BRE-TFIIB interaction as a
proposed mechanism of gene activation.
E
R
B
TFIIB
+1
Odd S. Gabrielsen
MBV4230
TFIIB-structure

C-terminal core domain (cTFIIB)





C-term core with two repeats (2x 75aa) that binds TBPTATA complex
each repeat = 5 α-helices → compact globular domain
(cyclin A-like)
HTH motiv that binds BRE (not conserved in yeast and
plants)
DNA-contact before and after TBP
C-term core
TFIIB
N
+1
N-terminal (nTFIIB) essential for
RNAPII contact


cysteine-rich region that forms a “zinc-ribbon” + B-finger
mediate contact wtih RNAPII-TFIIF complex through a
penetration mechanism
Odd S. Gabrielsen
MBV4230
TFIIBc structure
TBP
TS
S
Two globular repeats
contact DNA
before and after TBP
TFIIB
Odd S. Gabrielsen
MBV4230
Zn-ribbon + B-finger
= bridge to RNAPII
Odd S. Gabrielsen
MBV4230
TFIIB links TATA and RNAPII
and penetrates the active site
The C-terminal domain of TFIIB binds
the TBP-TATA one one side, and
contacts RNAPII on the other side.
TBP
BC link
TATA-pol
The N-terminal domain of TFIIB
(Zn ribbon) binds the dock
domain, where its B-finger
plunges down into the RNAPII
active center, loops back and
remerges across the saddle.
TFIIB
RNAPII
BN active
site
Odd S. Gabrielsen
MBV4230
TFIIB-B-finger penetrates RNAPII
Odd S. Gabrielsen
MBV4230
TFIIB-B-finger takes the place of RNA
Expelled when trx starts
B finger occupies the same location as
the DNA–RNA hybrid.
TFIIB may enhance the formation of an
early transcribing complex before a
length of 9 bp, required for optimal
stability, is attained.
As RNA grows, RNA and TFIIB must
compete for space. If RNA wins, TFIIB
is ejected and the pol is released from the
promoter to complete trx of the gene. If
TFIIB wins, initiation aborts and must be
tried again.
The B finger thus explains abortive
initiation and promoter escape.
TBP
BC link
TATA-pol
TFIIB
BN active
site
Odd S. Gabrielsen
MBV4230
Model for an RNAPII/IIF/IIB/TBP/DNA
Minimal Transcription Complex
Odd S. Gabrielsen
MBV4230
TFIIA

Controversial

not essensial in vitro with TBP and purified components
 required with TFIID and less purified system

Function

counteracts repressors associated with TFIID (Dr1, topoI, MOT1)
 Stabilizes the TBP-TFIIB complex
 TFIIA is able to enter the PIC assembly on all steps after TFIID
binding

Required for activator-response
Odd S. Gabrielsen
MBV4230
Structure of TFIIA

human/drosophila heterotrimer: 37 + 19 + 13 kDa (α, β, γ)


Both α and β product of the same gene - the αβ precursor is cleaved to α + β
yeast: heterodimer: 32 + 13 kDa


TOA1 32kDa (homologous to human α and β) essensial
TOA2 13 kDa essensial
Yeast TOA1
Human α

Antirepression requires β + γ

Activation requires α + β + γ

3D → two domains form an L-formed structure



Human ß
TOA1 and TOA2 intertwined
Both C-terminals generate a compact β-sheet (β -sandwich, β -barrel)
Both N-terminals generate a “four-helix bundle”
C
L
N
Odd S. Gabrielsen
MBV4230
TFIIA structure
C-terminal ß-barrel
contacts DNA and TBP
TFIIA
N-terminal
4-helix bundle.
Probably activator contact
Odd S. Gabrielsen
MBV4230
TFIIA structure
TBP
C-terminal ß-barrel
contacts DNA and TBP
TFIIA
N-terminal
4-helix bundle.
Probably Activator contact
Odd S. Gabrielsen
MBV4230
Yeast TFIIA + TBP + DNA
TBP
TFIIA
Odd S. Gabrielsen
MBV4230
TFIIA - DNA-interaction


Interaction with DNA upstream TATA
C-terminal β-barrel → both TBP- and DNA-interaction

TBP-TFIIA: the edges of the two β-structures interact → extended β-sheet

DNA-TFIIA: C-terminal β-barrel contacts phosphates 3 bp upstream TATA
Explains why TFIIA stabilizes TBP-DNA complex


TFIIAs N-terminal α-helix structure generates an interaction
domain necessary for activator contact



Rational explanation of:
Antirepression requires β + γ which generate β-barrel with TBP+DNA contact
Activation requires α + β + γ which also generate the N-terminal interaction
domain

TFIIA and TFIIB bind on opposite sides of DNA without
collision

TBPs convex surface still exposed for other interactions
Odd S. Gabrielsen
MBV4230
TFIIA-TBP-TFIIB: place for all
TBP
TFIIA
TFIIB
Odd S. Gabrielsen
MBV4230
TFIIF (also called RAP = RNAPII-ass. faktor)



Structure:

Heterodimer in higher eukaryotes: RAP30 + RAP74 (Mw: 26 + 58 kDa)

S.cer.TFIIF heterotrimer: 105, 54, 30 kDa
Distinct feature: function in initiation and elongation
Initiation - helps in the recruitment of RNAPII

TFII
Stable association of RNAPII requires TFIIF

TFIIF-TFIIB associate in solution
 TFIIF-RNAPII associate in solution
TFII

Initiation: a role in recruitment of TFIIE+TFIIH

Elongation: enhances catalytic velocity of RNAPII

More later
Odd S. Gabrielsen
MBV4230
TFIIF = heterotetramer (RAP302 RAP742)

RAP30: Two σ-related domains
RNAPII
DNA
TFIIB

RAP74:


Required for stimulation of elongation
RAP74 is strongly phosphorylated in vivo
 Kinase? Possibly TAFII250
 TFIIF becomes more active when phosphorylated
30 30
74
74
P
P P P
DNA
RNAPII
Odd S. Gabrielsen
MBV4230
TFIIF DNA-contacts


Complex pattern of protein-DNA
contacts
Explained by wrapping of DNA
around RNAPII-TFIIF?
Kornberg unpublished: TFIIF
binds the non-template DNA
strand
74

30
74
30
?
TATA
INR
Odd S. Gabrielsen
MBV4230
3D of TFIIF

TFIIF (blue) is distributed
across the surface of the
polymerase.

The distribution of the second
largest subunit of TFIIF is
very similar to the sigma
subunit of bacterial RNA
polymerase.
Odd S. Gabrielsen
MBV4230
Model of the RNAPII transcription
initiation complex
Odd S. Gabrielsen
MBV4230
TFIIE


Structure

heterotetramer α2β2: 56 + 34 kDa

Contacts DNA in and just downstream of trx bubble
34 34
Function in trx.initiation



TFIIEβ
Recruitment of TFIIH to PIC
Regulates the activity of TFIIH
Role in NER (nucleotide excision repair)




56
56
TFIIEα
Damage recognized by XPA
XPA binds TFIIE
TFIIE recruits TFIIH
Repairosome is formed
Odd S. Gabrielsen
MBV4230
TFIIH


The most complex of the GTFs - 9 subunits
The only GTF with enzymatic activity:

Two Helicases (ATP-dependent) Helicases are enzymes that catalyzes the



[ATPase (DNA-dependent)]
CTD-kinase
Kinase substrat:
separation of strands of a DNA double helix (or a
DNA-RNA hybrid) using the energy from ATP or
GTP hydrolysis. They move with a directionality
specific to each particular enzyme.

CTD - preferred substrate of Holo TFIIH
 GTFs




TBP
TFIIEα
TFIIFα (RAP74)
Andre TFs

Oct, p53, RARα, ERα, pRb
Odd S. Gabrielsen
MBV4230
TFIIH structure
Odd S. Gabrielsen
MBV4230
TFIIH-structure

Multisubunit factor ( human / yeast )










Helicases utilise the energy of nucleotide hydrolysis
to unwind nucleic acid duplexes.
NER - nucleotide excision repair
89 kDa XPB / SSL2 (p105) NER-function ATPase/3´-5´-helicase
 NTP-site mutated → lethal + trx.dead
 XPB-helicase is necessary for trx.activity
 Explains ATP requirement in initiation of trx
80 kDa XPD / RAD3 (p85) NER-function ATPase/5´-3´-helicase
 NTP-site mutated → not lethal + trx.OK + NER-defect
 XPD-helicase not required for trx. activity
62 kDa P62 / TFB1 (p75) UV-hypersens.
50 kDa P52 / TFB2 (p55)
44 kDa P44 / SSL1 (p50) (supr. of stem-loop) zinc finger motif
34 kDa P34 / TFB4 (p37) zinc finger motif
32 kDa MAT1 / TFB3 (p38) ring finger motif, cdk-assembly factor
38 kDa cyclin H / CCL1 (p45+p47) cyclin-partner for CDK7/MO15 and Kin28
40 kDa CDK7, MO15 / KIN28 (p32) cyclin-dependent kinase
Surprising
Link to
DNA-repair
core
kinase
TFIIH dual function: in transcription initiation and in NER
Odd S. Gabrielsen
MBV4230
Holo TFIIH = core TFIIH + CAK
linked by XPD
Bridge
Kinase
(CAK)
Core
CAK
Odd S. Gabrielsen
MBV4230
TFIIH multiple functions

Function 1: promoter-melting assisted by helicases (2
steps, see below)



Function 2: CTD-kinase, role in promoter clearance


Modell: CTD-phosphorylation after chain separation and initiation → PIC
disrupted → elongation complex leaves the promoter
Function 3: role in elongation


Model: 3´-5´-helicase + 5´-3´-helicase + ATP → chain separation around TSS
ATP-depent step in initation (in addition to CTD phosphorylation)
Model: TFIIH-kinase+ ATP → maintains hyperphosphorylated pol.II
(counteracting the CTD phosphatase)
Function 4: role in DNA-repair (NER)



5 of 9 subunits of TFIIH with a double function in trx.+repair
actively trx.genes are preferentially repaired
TFIIH can complement NER-deficient extract
Odd S. Gabrielsen
MBV4230
Assists in formation of open
complex and promoter escape
1. ATP-dependent promoter melting - chain separation - open
trx. complex
TFIIH helicase
2. ATP-dependent structural transition into an escapecompetent conformation
TFIIH helicase
Odd S. Gabrielsen
MBV4230
TFIIH: also linked to the cell cycle?

The TFIIH kinase = CAK = cdk7 + cyclin H + MAT-1




An open question :


CAK = CDK activating kinase (with a role in the cell cycle)
CAK activates other cdk´s through Thr-phosphorylation
MAT-1 (a ring-finger protein) makes CAK constitutively active (Thr-indep.)
Is CTD-phosphorylation regulated by the cell cycle?
Different answers :

No - probably not!




Argument : only 20% of all CAK in the cell is TFIIH-associated
Yeast has separate CAKs for TFIIH and cell cycle
Activity and level of CDK7, cyclin H and Mat1 do not change during cell cycle
Yes - May well be!



TFIIH inhibited during mitosis concomitant with inhibition of CDK7 (CDC2-induced)
Cell cycle inhibitor INK4 inhibits CTD phosphorylation by CDK7
CDK8 can negatively regulate CDK7
Odd S. Gabrielsen
MBV4230
Model
DNA repair
Transcription
Repairproteins
CAK
Core TFIIH
CAK
Core TFIIH
CAK
Cell cycle
Odd S. Gabrielsen
Sequential distortion
of DNA
MBV4230
PIC assembly - a gradual wrapping
process?
TB
TB
TFII RNAPI
TB
TFII RNAPI
TFII
TFII
Odd S. Gabrielsen
MBV4230
Topology model
Odd S. Gabrielsen
The trx cycle
MBV4230
Trx initiation and reinitiation
Odd S. Gabrielsen
Multiplicity of GTFs?
Are a single set of GTFs universally used?
…equally at all promoters?
MBV4230
Several GTF complexes possible

Several GTFs encoded by single copy genes



However, multiple genes exist for specific GTFs




TFIIB, E, F, and H
Also true for RNAPII
Multiple TFIIA related
Multiple TFIID related
Gene-selective developmental roles?
Consequence: several possible complexes possible


By replacing ”normal” versions with specific ones
By generating variant combinations of GTF-containing complexes
Odd S. Gabrielsen
MBV4230
Variant TBPs:
TRFs = TBP related factors
≥2 TBP like proteins
in multicellular organisms
TBP top view
Drosophila
TRF1 ≈
TBP
TRF2
TLP
TLF
TRF
TRP
TBP bottom view
• TRF1 - major part of TFIIIB, a RNAPIII factor
• TRF1 binds pref TC-box (TTTTCT) in the core
promoter of the Drosophila tudor gene, a direct target
TBP specific
TRF2 specific
Odd S. Gabrielsen
MBV4230
A diversity of complexes



Many TBP
complexes
Alternative TAFcontaining
complexes
Variant TFIIAs
Odd S. Gabrielsen
MBV4230
A diversity of core promoters may
assemble gene-specific complexes

TATA core promoters require TBP, but not necessarily
TAFs

Inr ± DPE core promoters require TAFs and hence
indirectly TBP associated
TLF-dependent core promoters do not require TBP

Odd S. Gabrielsen
MBV4230
Diversity of core promoters



GTF machinery shows some diversity
Activators and repressors (Tfs) show enormous
diversity
Not thousands to one, but thousands to several
Enormous
diversity
Some
diversity
Odd S. Gabrielsen
MBV4230
Examples of questions for the exam

TFIIH
 One
of the GTFs (general transcription factors) has enzymatic
activities – which GTF and what type of enzymatic activity?

TFIIB
 RNAPII
cooperates with general transcription factors (GTFs)
to form a functional pre-initiation complex (PIC). Describe
how the GTF called TFIIB operates during PIC assembly. In
particular, point out how TFIIB interacts with promoter DNA,
with other GTFs and with RNAPII and try to provide a
functional explanation for the interactions where relevant.
51
Odd S. Gabrielsen