Transcript Document

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
activator interference or squelching
activator B
activator A
UAS
hypothesis?
TATA box
what is the limiting target of activators?
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’
Isolation of coactivators associated with the TATA-binding
protein that mediate transcriptional activation
Dynlacht, Hoey, Tjian, Cell 1991
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Robert Tjian
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 activation
Dynlacht, 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 activation
Dynlacht, 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 activation
Dynlacht, 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
RNA Polymerase II Transcription Machinery
Number of subunits
Pol II
12
GTFs
TFIID
TFIIB
TFIIE
TFIIH
TFIIF
TFIIA*
Mediator
TBP
TAFs *
1
12
1
2
9
2
3
22
Assembly of
recombinant TFIID
reveals differential
coactivator
requirements for
distinct transcriptional
activators
Chen 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 activators
Chen et al., Cell 1994
Assembly of recombinant TFIID reveals differential coactivator
requirements for distinct transcriptional activators
Chen 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 activators
Chen 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
activators
Chen et al., Cell 1994
The ‘TFIID hypothesis’
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1. TAFs provide surfaces for the interaction
of TFIID with activators.
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2. TFIID recruits polymerase
in vitro assays suggest specific activator-TAF contacts
predictions?
Yeast TAFII145 functions as a core promoter selectivity
factor, not a general coactivator
Walker et al., Cell, 1997
Shen 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 coactivator
Walker et al., Cell, 1997
Shen 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.
What is the Limiting Target of Activators?
activator B
activator A
UAS
TATA box
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)
Gal4-VP16
TATA
X
UAS(dA-dT)2
UASGAL10
autoinhibition
TATA
activator
interference
UASGAL10
UAS(dA-dT)2
A novel mediator between activator proteins and the RNA
polymerase II transcription apparatus (Kelleher et al. 1990)
Gal4-VP16
Yeast
nuclear extract
in 50 mM (NH4)2SO4
X
UAS(dA-dT)2
TATA
400 mM Elu
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TIFF ( LZW) dec ompr ess or
ar e n eed ed to s ee thi s pi ctu re.
DEAE column
50 mM
50 mM FT
400 mM (NH4)2SO4
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)
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
Squelching in vitro: interpretation
autoinhibition
UASG
TATA
hypothetical target of activators
UASG
TATA
excess
Gal4-VP16
activator
interference
UASdA-dTTATA
UASdA-dTTATA
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
A mediator required for activation of RNA polymerase II
transcription in vitro Flanagan et al., Nature, 1991
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
Gcn4 squelches
Gal4-VP16
Gal4-VP16
squelches Gcn4
squelching is observed with the mediator fraction
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
RNA polymerase II Transcription Machinery
Number of subunits
Pol II
12
GTFs
TFIID
TFIIB
TFIIE
TFIIH
TFIIF
TFIIA*
Mediator
TBP
TAFs *
1
12
1
2
9
2
3
22
Crystal Structure of Yeast RNA Polymerase II at 2.8 Å
Resolution (Cramer et al, 2001)
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CTD
the carboxy-terminal domain (CTD) of RNA polymerase II
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CTD facts:
- unique to RNA pol II
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Heptapeptide of the CTD
(52 repeats in mammalian Rpb1, 27 in yeast)
- 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 antibody
separates mediator from
RNA polymerase II
An RNA polymerase II holoenzyme responsive to activators
Koleske 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 activators
Koleske 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
holoenzyme supports activator
dependent transcription in vitro
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
TFIIH phosphorylates
Ser5 at initiation
P-TEFb phosphorylates Ser2
during elongation
Ser5-P
Ser2-P
CTD phosphorylation:
Pre-initiation
initiation/escape
elongation
a cycle of CTD modification during transcription
stage in transcription cycle
pre-initiation
early elongation
phospho Ser 2, 5
later elongation
CTD repeat=YS2PTS5PS
the CTD phosphorylation cycle coordinates diverse events
during transcription
mediator
stage in transcription cycle
pre-initiation
capping enzyme
early elongation
later elongation
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. extract
5x G4-act
wash
Holoenzyme Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Mediator
Holoenyme
TFIIE
TFIIF
Pol II
TFIIH
activator
TBP
TFIIB
PIC
Mediator Bound to RNA Polymerase II
(Single Particle analysis)
Clamp
Tail
F. Asturias
Srb2
Srb4
Head
Srb5
Head Srb6
Med6 Rgr1
Med8 Srb7
Med11 Med1
Middle
Med4
Middle Med7
Rox3
Nut1
Sin4
Nut2
Med2
Tail
Cse2
Med3
Gal11
2 conformations of mediator
Holoenzyme Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Mediator
Holoenyme
TFIIE
TFIIF
Pol II
TFIIH
activator
TBP
TFIIB
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 Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators
recruit polymerase to the promoter.
Mediator
Holoenyme
TFIIE
TFIIF
Pol II
TFIIH
activator
TBP
TFIIB
PIC
predictions of model?
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
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Association of the Mediator complex with enhancers of
active genes Kuras et al., PNAS 2003
Mediator
Binding
mediator binding did not
depend on pol II binding or
the TATA boxes
TATA
TATA
GAL10
enhancer
GAL1
The Swi5 activator recruits
the Mediator complex to the
HO promoter without RNA
polymerase II Bhoite et al.,
Genes & Dev., 2001
Mediator can be
recruited to genes
independently of Pol II,
GTFs and transcription
-often, recruitment is to
enhancers rather than
core promoter
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 factor
2. temp. shift experiment show that it is required prior to
initiation
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Mediator as a general transcription factor
Takagi and Kornberg, JBC, 2005
Srb4ts
E(30°C)
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
Some TAFs function in promoter recognition
TFIIB
TBP
TAFs
TATA
TAFs
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Inr
DPE
(Verrijzer et al, 1995)
New Model: TAFs function in core promoter recognition
Are all TAFs devoted to
promoter recognition?
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Goodrich et al, (1996)
TBP and TAF homologs may mediate tissue specific
gene expression patterns in differentiated cells
TRF=TBP related factor
Reina JH, Hernandez N. Genes Dev. 2007