Recap eukaryotes have 3 nuclear RNA polymerases, which transcribe unique sets of genes RNA pol II transcribes protein coding genes and must respond to

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

TATA box

activator A

UAS

activator B

hypothesis?

activator interference or squelching

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?

QuickTime™ and a decompressor

are needed to see this picture.

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


QuickTime™ and a decompressor


recombinant TBP cannot substitute for TFIID when assaying activated transcription in vitro


TBP, the TATA-binding protein is small, but glycerol gradient sedimentation and gel filtration chromatography indicates that TFIID is very large


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



TAFII150 and TAFII60 are sufficient for activation by NTF-1


NTF-1 activation domain peptide on beads

TAFII150 and TAFII60 are specifically retained


QuickTime™ and aTIFF (LZW) decompressor


The ‘TFIID hypothesis’

in vitro assays suggest specific activator-TAF contacts

predictions?





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.

TATA box

activator A

UAS

activator B

What is the Limiting Target of Activators?

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

QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.

DEAE column

the 400 mM fraction overcomes squelching by Gal4-VP16



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


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


squelching is observed with the mediator fraction


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*

*

QuickTime™ and aYUV420 codec decompressor


Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001)

CTD





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 by E1A and Elk1 in vitro requires Sur2

no effect on activation by VP16


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

2 conformations of mediator



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



Holoenyme

activator

Mediator

Pol IITFIIF

TBP

TFIIB

TFIIH

TFIIE

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

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



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”




1. mediator behaves like a general transcription factor2. temp. shift experiment show that it is required prior to initiation

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

TATA

Inr

DPE



TAFs TAFs QuickTime™ and aTIFF (LZW) decompressor


TBPTFIIB

Some TAFs function in promoter recognition

(Verrijzer et al, 1995)



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

Documents

Recap eukaryotes have 3 nuclear RNA polymerases, which transcribe unique sets of genes RNA pol II transcribes protein coding genes and must respond to