1. 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

2. activator interference or squelching
activator B
activator A
UAS
TATA box
hypothesis?

3. ‘Holoenzyme hypothesis’
what is the limiting target of activators?
Eukaryotic activators do not bind to RNA pol II polymerase and therefore do not directly recruit polymerase to promoters.
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’

4. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation
Dynlacht, Hoey, Tjian, Cell 1991
Robert Tjian
in 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

5. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation
Dynlacht, Hoey, Tjian, Cell 1991
recombinant TBP cannot substitute for TFIID when assaying activated transcription in vitro

6. 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

7. 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

8. RNA Polymerase II Transcription Machinery
Number of subunits
Pol II 12
GTFs
TFIID
TFIIB 1
TFIIE 2
TFIIH 9
TFIIF 2
TFIIA 3
Mediator 22
TBP *
TAFs
Much attention has been focused on the TFIID complex, since TFIID is critical for preinitiation complex formation and provides many interacting surfaces due to its potentially large number of subunits. *

9. 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

10. Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators
Chen et al., Cell 1994

11. 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

12. 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

13. Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators
Chen et al., Cell 1994

14. The ‘TFIID hypothesis’
1. TAFs provide surfaces for the interaction of TFIID with activators.
2. TFIID recruits polymerase
Note that, contrary to the apparent lack of specificity of activation domains, the TFIID hypothesis presumes specific interactions between activators and TAFs.
in vitro assays suggest specific activator-TAF contacts
predictions?

15. 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

16. 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!

17. 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

18. Conclusions:
Several activators can activate transcription in vitro in the absence of TAFs.
Not all transcription depends on TAFs in vivo.
(based on analysis of yeast TAF mutants)
Some TAFs may assist in recognition of the core promoter (rather than transmitting regulatory information associated with upstream factors).
TAFs and alternative TBPs may specify selection of particular core promoters.

19. What is the Limiting Target of Activators?
activator B
activator A
UAS
TATA box

20. A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)

21. A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
Gal4-VP16 X
TATA
UASGAL10
UAS(dA-dT)2
TATA
autoinhibition
activator interference
UASGAL10
UAS(dA-dT)2

22. A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990)
Gal4-VP16 X
Yeast
nuclear extract
in 50 mM (NH4)2SO4
UAS(dA-dT)2
TATA
400 mM Elu
DEAE column
50 mM FT
50 mM
400 mM
(NH4)2SO4
the 400 mM fraction overcomes squelching by Gal4-VP16

23. 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

24. 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

25. Squelching in vitro: interpretation
TATA
UASG
TATA
UASG
autoinhibition
excess Gal4-VP16
hypothetical target of activators
activator interference
TATA
UASdA-dT
TATA
UASdA-dT

26. 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

27. 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

28. 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

29. 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

30. 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

31. RNA polymerase II Transcription Machinery
Number of subunits
Pol II 12
GTFs
TFIID
TFIIB 1
TFIIE 2
TFIIH 9
TFIIF 2
TFIIA 3
Mediator 22
TBP *
TAFs *

32. Crystal Structure of Yeast RNA Polymerase II at 2
Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001)
An important component of RNA pol II is not revealed in the crystal structure, presumably because it is mostly if not entirely unstructure: the rather large carboxy-terminal domain of Rpb1. The site of its attachment to the enzyme is indicated.
CTD

33. 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
Upon initiation of transcription the CTD heptapeptides are phosphorylated at 5 by the kinase subunit of TFIIH . Later, Ser5 -P levels decline, and Ser2 is phosphorylated by P-Tefb, which as I mentioned last week, plays a role in regulation of elongation.
Heptapeptide of the CTD
(52 repeats in mammalian Rpb1, 27 in yeast)

34. 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

35. 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

36. 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

37. 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

38. 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
purified mediator binds the CTD in vitro

39. the CTD is required for mediator activated transcription 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

40. 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
Upon initiation of transcription the CTD heptapeptides are phosphorylated at 5 by the kinase subunit of TFIIH . Later, Ser5 -P levels decline, and Ser2 is phosphorylated by P-Tefb, which as I mentioned last week, plays a role in regulation of elongation.

41. a cycle of CTD modification during transcription
stage in transcription cycle
pre-initiation
early elongation
CTD repeat=YS2PTS5PS
phospho Ser 2, 5
later elongation

42. splicing and polyA factors
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

43. 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

44. 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

45. 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

46. 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

47. Holoenzyme Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol II
TFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC
The notion of a holoenzyme lent support to the recruitment hypothesis. In this model Mediator simply functions as a handle to allow recruitment of Pol II to promoters. Activators interact with Mediator, which exists in complex with Pol II. Eukaryotic gene regulation appeared to follow the same principals as the prokaryotic paradigm of CAP function at the lac operon.

48. Mediator Bound to RNA Polymerase II
(Single Particle analysis)
Srb2
Srb4
Srb5
Srb6
Med6
Med8
Med11
Head
Head
Clamp
Rgr1
Srb7
Med1
Med4
Med7
Rox3
Nut1
Nut2
Cse2
Middle
Middle
Sin4
Med2
Med3
Gal11
Tail
Tail
F. Asturias

49. 2 conformations of mediator

50. Holoenzyme Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol II
TFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC
The notion of a holoenzyme lent support to the recruitment hypothesis. In this model Mediator simply functions as a handle to allow recruitment of Pol II to promoters. Activators interact with Mediator, which exists in complex with Pol II. Eukaryotic gene regulation appeared to follow the same principals as the prokaryotic paradigm of CAP function at the lac operon.

51. 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

52. Holoenzyme Hypothesis
Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter.
Holoenyme
activator
Mediator
Pol II
TFIIF
TBP
TFIIB
TFIIH
TFIIE
PIC
The notion of a holoenzyme lent support to the recruitment hypothesis. In this model Mediator simply functions as a handle to allow recruitment of Pol II to promoters. Activators interact with Mediator, which exists in complex with Pol II. Eukaryotic gene regulation appeared to follow the same principals as the prokaryotic paradigm of CAP function at the lac operon.
predictions of model?

53. recruitment of the mediator is sufficient for activated transcription
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
The result of the activator-bypass experiment is consistent with the recruitment model. However, LexA-Gal11 may as well recruit other factors rather than mediator. Since this possibility has not been excluded in any of the bypass experiments, they do not amount to a convincing argument after all.

54. mediator binding did not depend on pol II binding or the TATA boxes
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
GAL10
GAL1
enhancer
TATA
Mediator
Binding

55. 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

56. 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”
If Mediator operates by a recruitment mechanism alone it should be possible to dispense with the requirement for Mediator by increasing the concentration of Pol II and GTFs to increase promoter occupancy by the transcription machinery. The following experiment supports the notion that Mediator operates at a post-recruitment step.

57. 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
If Mediator operates by a recruitment mechanism alone it should be possible to dispense with the requirement for Mediator by increasing the concentration of Pol II and GTFs to increase promoter occupancy by the transcription machinery. The following experiment supports the notion that Mediator operates at a post-recruitment step.

58. Mediator as a general transcription factor
Takagi and Kornberg, JBC, 2005
Srb4ts E(30°C)
If Mediator operates by a recruitment mechanism alone it should be possible to dispense with the requirement for Mediator by increasing the concentration of Pol II and GTFs to increase promoter occupancy by the transcription machinery. The following experiment supports the notion that Mediator operates at a post-recruitment step.
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

59. Some TAFs function in promoter recognition
TATA
TFIIB
TBP
TAFs
TAFs
Inr
TAF1 stimulates TBP binding not by providing a bridge between an activator and TBP, but by making contact with promoter DNA itself.
DPE
(Verrijzer et al, 1995)

60. New Model: TAFs function in core promoter recognition
Are all TAFs devoted to promoter recognition?
Goodrich et al, (1996)

61. TRF=TBP related factor
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