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Recap
•eukaryotes have 3 nuclear RNA polymerases, which transcribe unique sets of genes
•RNA pol II transcribes protein coding genes and must respond to and integrate a diverse set of signals in order to regulate expression of >25k genes
•in vitro transcription systems for pol II show accurate initiation
•gene specific regulators in euks have separable DNA binding and activation domains, the role of the DNA binding domain is to tether the activation domain near the promoter
•activation domains have no clear distinguishing structural or sequence features that indicate their mechanism of action
•squelching experiments indicate that activators compete for some limiting factor (not the polymerase)
•TFIID and holoenzyme hypotheses may explain activator function
1. Eukaryotic activators do not bind to RNA pol II polymerase and therefore do not directly recruit polymerase to promoters.
2. Activators may, however, indirectly recruit RNA polymerase by recruiting factors (often called co-activators) that serve as a physical bridge between activator and polymerase.
‘TFIID hypothesis’
‘Holoenzyme hypothesis’
what is the limiting target of activators?
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Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activationDynlacht, Hoey, Tjian, Cell 1991
Robert Tjianin vitro transcription reactions assembled from partially purified basal transcription factors
-pol II ~90% pure-general factors <1% pure
when assaying basal transcription (no activator present) in vitro, recombinant TBP can substitute for TFIID
Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activationDynlacht, Hoey, Tjian, Cell 1991
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recombinant TBP cannot substitute for TFIID when assaying activated transcription in vitro
Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activationDynlacht, Hoey, Tjian, Cell 1991
TBP, the TATA-binding protein is small, but glycerol gradient sedimentation and gel filtration chromatography indicates that TFIID is very large
Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activationDynlacht, Hoey, Tjian, Cell 1991
the coactivator activity can be separated from TBP by ion exchange chromatography of TFIID in the presence of urea
TBP and TBP-associated factors are required for activated transcription
Pol II 12
GTFs TFIID
TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3
Mediator 22
RNA Polymerase II Transcription MachineryNumber of subunits
TBP 1TAFs 12*
*
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activatorsChen et al., Cell 1994
had cloned and expressed most of the TAFs
worked out methods for reconstitution of complex entirely from recombinant proteins
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activatorsChen et al., Cell 1994
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activatorsChen et al., Cell 1994
TAFII150 and TAFII60 are sufficient for activation by NTF-1
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activatorsChen et al., Cell 1994
NTF-1 activation domain peptide on beads
TAFII150 and TAFII60 are specifically retained
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activatorsChen et al., Cell 1994
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The ‘TFIID hypothesis’
in vitro assays suggest specific activator-TAF contacts
predictions?
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1. TAFs provide surfaces for the interaction
of TFIID with activators. 2. TFIID recruits polymerase
Yeast TAFII145 functions as a core promoter selectivity factor, not a general coactivatorWalker et al., Cell, 1997Shen and Green, Cell, 1997
polyA+ RNA levels are largely unaffected by inactivation of TAFs
cell cycle regulated genes appear to be TAF-dependent
Yeast TAFII145 functions as a core promoter selectivity factor, not a general coactivatorWalker et al., Cell, 1997Shen and Green, Cell, 1997
TAFII145 dependence tracks with the core promoter, not the UAS!
Transcriptional activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs Oelgeschlager et al., 1998
western blot demonstrating depletion of TAFIIs
in vitro transcription shows that - transcription is abolished in the TFIID depleted extract- TBP is sufficient to restore activated transcription- 4 different activators were tested
no transcription after depletion of TFIID and TAFs
Conclusions:
1. Several activators can activate transcription in vitro in the absence
of TAFs.
2. Not all transcription depends on TAFs in vivo.
(based on analysis of yeast TAF mutants)
3. Some TAFs may assist in recognition of the core promoter (rather
than transmitting regulatory information associated with upstream
factors).
4. TAFs and alternative TBPs may specify selection of particular core
promoters.
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
UASGAL10
UAS(dA-dT)2
TATAUAS(dA-dT)2
X
TATA UASGAL10
Gal4-VP16
autoinhibition activator interference
Yeastnuclear extractin 50 mM (NH4)2SO4
50 mM 400 mM (NH4)2SO4
50 mM FT
400 mM Elu
TATAUAS(dA-dT)2
XGal4-VP16
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DEAE column
the 400 mM fraction overcomes squelching by Gal4-VP16
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
Potential explanations?
-column fraction has activator for the template
-something in column is binding/sequestering Gal4-VP16
-general stimulatory effect
-fraction contains some limiting basal factor
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
Potential explanations?
-column fraction has activator for the template-no, it doesn’t squelch a Gal4 template
-something in column is binding/sequestering Gal4-VP16-no, activation by Gal4-VP16 is not disrupted
-general stimulatory effect-no, activation depends upon Gal4-VP16
-fraction contains some limiting basal factor-no, adding them back does not overcome squelching
TATAUASG
autoinhibition
activator interference
TATAUASdA-dT
Squelching in vitro: interpretation
TATAUASG
TATAUASdA-dT
excess Gal4-VP16hypothetical target of activators
A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991
response to activators is lost during purification of general factors, but basal transcription (0ug Gal4-VP16) is unchanged
mediator fraction restores activator response in a purified in vitro transcription system
general transcription factors do not have mediator activity
A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991
squelching is observed with the mediator fraction
A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991
Gcn4 squelches Gal4-VP16
Gal4-VP16squelches Gcn4
A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994) -Srb5 IP
A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994)
components of holo-RNA polymerase II:-12 polymerase subunits-3 TFIIF subunits-SRB proteins-Gal11, Sug1
Pol II 12
GTFs TFIID
TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3
Mediator 22
RNA polymerase II Transcription MachineryNumber of subunits
TBP 1TAFs 12*
*
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Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001)
CTD
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Heptapeptide of the CTD(52 repeats in mammalian Rpb1, 27 in yeast)
the carboxy-terminal domain (CTD) of RNA polymerase II
CTD facts:
- unique to RNA pol II
- the CTD is not required for transcription in vitro
-the CTD is essential for life
-the CTD is subject to a cycle of phosphorylation at serines 2 and 5
- may be simultaneously phosphorylated at Ser2,5
A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994)
an anti-CTD antibodyseparates mediator from RNA polymerase II
An RNA polymerase II holoenzyme responsive to activatorsKoleske and Young, Nature, 1994
previously: •CTD truncation mutations limit the response to activators
•isolated srb mutation as suppressors of CTD truncation mutations in yeast
•purified a complex of Srb proteins with pol II and basal factors “RNA polymerase II holoenzyme”
Srb proteins copurify with RNA pol II
An RNA polymerase II holoenzyme responsive to activatorsKoleske and Young, Nature, 1994
previously: •CTD truncation mutations limit the response to activators
•isolated srb mutation as suppressors of CTD truncation mutations in yeast
•purified a complex of Srb proteins with pol II and basal factors “RNA polymerase II holoenzyme”
holoenzyme supports activatordependent transcription in vitro
Srb proteins copurify with RNA pol II
Transcriptional activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs Oelgeschlager et al., 1998
depletion of Srb7
decreased transcription response to activator
The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain Myers et al., Genes & Dev., 1998
purified mediator binds the CTD in vitro
The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain Myers et al., Genes & Dev., 1998
the CTD is required for mediator activated transcription in vitro
a cycle of CTD modification during transcription
Ser5-P Ser2-P
CTD phosphorylation:
Pre-initiation initiation/escape elongation
TFIIH phosphorylates Ser5 at initiation
P-TEFb phosphorylates Ser2 during elongation
CTD repeat=YS2PTS5PS
phospho Ser 2, 5
a cycle of CTD modification during transcription
pre-initiation
early elongation
later elongation
stage in transcription cycle
early elongation
the CTD phosphorylation cycle coordinates diverse events during transcription
pre-initiation
later elongation
stage in transcription cyclemediator
capping enzyme
splicing and polyA factors
Association of an activator with an RNA polymerase II holoenzyme Hengartner et al., 1995, Genes & Dev.
holoenzyme is retained on a GST-VP16 column
a mutation that abolishes activation by VP16 also abolishes holoenzyme binding
Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003
adenovirus E1A protein activates transcription of early genes by pol II
E1A binds the Sur2 subunit of mediator in vitro and associates with mediator in vivo
E1A mutations that prevent activation also disrupt Sur2 binding
sur2-/- ES cells:-all other mediator subunits still in the complex-E1A doesn’t activate-several other activators still work
Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003
activation by E1A and Elk1 in vitro requires Sur2
no effect on activation by VP16
Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003
Sur2 is required for binding of mediator and GTFs to E1A-bound promoters
Nuc. extract5x G4-act
wash
Holoenzyme HypothesisMediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol IITFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC
Rgr1Srb7Med1Med4Med7Rox3Nut1Nut2Cse2
Middle
Srb2Srb4Srb5Srb6Med6Med8Med11
Head
Sin4Med2Med3Gal11
Tail
Mediator Bound to RNA Polymerase II(Single Particle analysis)
F. Asturias
Clamp
Head
Middle
Tail
Holoenzyme HypothesisMediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol IITFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC
mediator summary
1. Of the 20 mediator subunits in yeast, 13 had been identified previously in genetic screens for factors affecting transcription.
2. 11 mediator subunits are essential for life
3. Mediator appears to required for all pol II transcription (a general factor?)
4. Homologs for almost all Mediator subunits observed in fungi, plant and metazoan genomes.
5. Strong structural similarity observed between mediator complexes of yeast, mice, humans
6. In some cases, activators have been shown to contact specific mediator subunits and disruption of these contacts disrupts transcription
Holoenzyme HypothesisMediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol IITFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC predictions of model?
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Gene activation by recruitment of the RNA polymerase II holoenzyme Farrel et al., Genes and Dev., 1996
recruitment of the mediator is sufficient for activated transcription
GAL10 GAL1enhancer
TATATATA
Med
iato
r B
indi
ng
Association of the Mediator complex with enhancers of active genes Kuras et al., PNAS 2003
mediator binding did not depend on pol II binding or the TATA boxes
Mediator can be recruited to genes independently of Pol II, GTFs and transcription
-often, recruitment is to enhancers rather than core promoter
The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II Bhoite et al., Genes & Dev., 2001
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Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005
purified mediator from WT and srb4ts strains
performed in vitro transcription reactions in the absence of activators “basal transcription”
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Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005
1. mediator behaves like a general transcription factor2. temp. shift experiment show that it is required prior to initiation
Srb4ts E(30°C)
Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005
excess RNA pol II or basal factors cannot complement the transcription defect of the srb4ts mediator preparation
suggests that mediator can act after recruitment of Pol II and general factors
TATA
Inr
DPE
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TAFs TAFs QuickTime™ and aTIFF (LZW) decompressor
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TBPTFIIB
Some TAFs function in promoter recognition
(Verrijzer et al, 1995)
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New Model: TAFs function in core promoter recognition
Goodrich et al, (1996)
Are all TAFs devoted to promoter recognition?
TBP and TAF homologs may mediate tissue specific gene expression patterns in differentiated cells
TRF=TBP related factorReina JH, Hernandez N. Genes Dev. 2007