GTFs and PIC assembly - T TB
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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
TFII
+ TFII
= PIC
Highly conserved TFII
Correct
initiation
RNAPII+GTFs = ca. 30 polypeptides of trx
TFII
≈ 2 MDa 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 Other factors
recognition event during PIC assembly
Binds a variety of different TATA-like sequences
A slow binding reaction N
minor groove contact DNA
binds as monomer
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
Carries all essential functions
TFIID
N-term domain divergent
probably involved in regulating DNA binding
TBP
N
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 .. but this way
this
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 TBP-
TATA box complex.
TFIIB can directly recognize
the unbent and/or bent TBP-
TATA-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 TAT
interaction also with TFIIF A
BRE
TFIIB +1
TSS
Function in initiation: “Measures” distance TATA - TSS
Odd S. GabrielsenMBV4230
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.
R E
B
TFIIB
+1
Odd S. GabrielsenMBV4230
TFIIB-structure
C-terminal core domain (cTFIIB)
C-term core with two repeats (2x 75aa) that binds TBP-
TATA complex
each repeat = 5 α-helices → compact globular domain
(cyclin A-like)
HTH motiv that binds BRE (not conserved in yeast and C-term core
plants)
DNA-contact before and after TBP TFIIB +1
N
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. GabrielsenMBV4230
TFIIBc structure
TBP
Two globular repeats
contact DNA
TS
S before and after TBP
TFIIB
Odd S. GabrielsenMBV4230
Zn-ribbon + B-finger
= bridge to RNAPII
Odd S. GabrielsenMBV4230
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 TFIIB
(Zn ribbon) binds the dock
domain, where its B-finger BN active
site
plunges down into the RNAPII RNAPII
active center, loops back and
remerges across the saddle.
Odd S. GabrielsenMBV4230
TFIIB-B-finger penetrates RNAPII
Odd S. GabrielsenMBV4230
TFIIB-B-finger takes the place of RNA
Expelled when trx starts
B finger occupies the same location as
the DNA–RNA hybrid.
TBP
TFIIB may enhance the formation of an BC link
early transcribing complex before a TATA-pol
length of 9 bp, required for optimal
stability, is attained.
As RNA grows, RNA and TFIIB must
compete for space. If RNA wins, TFIIB TFIIB
is ejected and the pol is released from the
promoter to complete trx of the gene. If BN active
site
TFIIB wins, initiation aborts and must be
tried again.
The B finger thus explains abortive
initiation and promoter escape.
Odd S. GabrielsenMBV4230
Model for an RNAPII/IIF/IIB/TBP/DNA
Minimal Transcription Complex
Odd S. GabrielsenMBV4230
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. GabrielsenMBV4230
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 Yeast TOA1
TOA2 13 kDa essensial
Human α
Antirepression requires β + γ Human ß
Activation requires α + β + γ
3D → two domains form an L-formed structure
TOA1 and TOA2 intertwined C
Both C-terminals generate a compact β-sheet (β -sandwich, β -barrel)
Both N-terminals generate a “four-helix bundle” L N
Odd S. GabrielsenMBV4230
TFIIA structure
C-terminal ß-barrel
contacts DNA and TBP TFIIA
N-terminal
4-helix bundle.
Probably activator contact
Odd S. GabrielsenMBV4230
TFIIA structure
TBP
C-terminal ß-barrel
contacts DNA and TBP TFIIA
N-terminal
4-helix bundle.
Probably Activator contact
Odd S. GabrielsenMBV4230
Yeast TFIIA + TBP + DNA
TBP
TFIIA
Odd S. GabrielsenMBV4230
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. GabrielsenMBV4230
TFIIA-TBP-TFIIB: place for all
TBP
TFIIA
TFIIB
Odd S. GabrielsenMBV4230
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
Stable association of RNAPII requires TFIIF
TFII
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. GabrielsenMBV4230
TFIIF = heterotetramer (RAP302 RAP742)
RNAPII
RAP30: Two σ-related domains
DNA
30 30
TFIIB
RAP74:
Required for stimulation of elongation
74 74
P
RAP74 is strongly phosphorylated in vivo
P P P
Kinase? Possibly TAFII250
TFIIF becomes more active when phosphorylated DNA
RNAPII
Odd S. GabrielsenMBV4230
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. GabrielsenMBV4230
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. GabrielsenMBV4230
Model of the RNAPII transcription
initiation complex
Odd S. GabrielsenMBV4230
TFIIE
TFIIEβ
Structure
heterotetramer α2β2: 56 + 34 kDa
34 34
Contacts DNA in and just downstream of trx bubble
Function in trx.initiation
Recruitment of TFIIH to PIC 56 56
Regulates the activity of TFIIH
Role in NER (nucleotide excision repair) TFIIEα
Damage recognized by XPA
XPA binds TFIIE
TFIIE recruits TFIIH
Repairosome is formed
Odd S. GabrielsenMBV4230
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)] separation of strands of a DNA double helix (or a
DNA-RNA hybrid) using the energy from ATP or
CTD-kinase GTP hydrolysis. They move with a directionality
Kinase substrat: 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. GabrielsenMBV4230
TFIIH structure
Odd S. GabrielsenMBV4230
Helicases utilise the energy of nucleotide hydrolysis
TFIIH-structure
to unwind nucleic acid duplexes.
NER - nucleotide excision repair
Multisubunit factor ( human / yeast ) Surprising
89 kDa XPB / SSL2 (p105) NER-function ATPase/3´-5´-helicase Link to
NTP-site mutated → lethal + trx.dead
DNA-repair
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 core
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 kinase
TFIIH dual function: in transcription initiation and in NER
Odd S. GabrielsenMBV4230
Holo TFIIH = core TFIIH + CAK
linked by XPD
Core
Bridge
Kinase
(CAK) CAK
Odd S. GabrielsenMBV4230
TFIIH multiple functions
Function 1: promoter-melting assisted by helicases (2
steps, see below)
Model: 3´-5´-helicase + 5´-3´-helicase + ATP → chain separation around TSS
ATP-depent step in initation (in addition to CTD phosphorylation)
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: 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. GabrielsenMBV4230
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 escape-
competent conformation
TFIIH helicase
Odd S. GabrielsenMBV4230
TFIIH: also linked to the cell cycle?
The TFIIH kinase = CAK = cdk7 + cyclin H + MAT-1
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.)
An open question :
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. GabrielsenMBV4230
Model
Transcription DNA repair
Repair-
proteins
CAK CAK
Core TFIIH Core TFIIH
CAK
Cell cycle
Odd S. GabrielsenSequential distortion of DNA
MBV4230
PIC assembly - a gradual wrapping
process?
TB
TB
TFII RNAPI
TB
TFII RNAPI
TFII
TFII
Odd S. GabrielsenMBV4230
Topology model
Odd S. GabrielsenThe trx cycle
MBV4230
Trx initiation and reinitiation
Odd S. GabrielsenMultiplicity 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
TFIIB, E, F, and H
Also true for RNAPII
However, multiple genes exist for specific GTFs
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. GabrielsenMBV4230
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. GabrielsenMBV4230
A diversity of complexes
Many TBP
complexes
Alternative TAF-
containing
complexes
Variant TFIIAs
Odd S. GabrielsenMBV4230
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. GabrielsenMBV4230
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. GabrielsenMBV4230
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
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