Building the preinitiation complex

1
Transcriptional regulation in Eukaryotes Part
1 Genes VIII Chapters 21-22 skip sections
21.19-21.25 Genes VII Chapters 20-21
2
Building the preinitiation complex (PIC)

• TFIID (800 kD larger than RNA pol II at 500
  kD)

TBP TATAA binding protein TAF TBP associated
factor
TAFs (9 to14)
TBP
mRNA
TATAAA
3
Basal factors

• TATA binding protein (TBP) 30 Kd
• 1. Binds minor groove of DNA.

N-
C-
View from 1 back upstream at TBP bound at TATAAA
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Basal factors

• TBP (continued)
• 2. TBP is one of the elements that determine the
  polarity of transcription. It is based on the
  asymmetry in the TATAAA sequence itself.

C-
transcription
N-
TATAAA
-Steve Burleys lab
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TBP induces a kink in the DNA
side view
TBP
80º bend
TATAAAAG
kinks result from insertion of 2 phenyalanines
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Basal factors

• TATA Binding Protein (TBP)

concave surface in contact with minor groove of
DNA
N-term
H2
H1
stirrup
stirrup
DNA
View upstream looking into plane towards 1
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Basal factors

• TATA Binding Protein (TBP)

concave surface in contact with minor groove of
DNA
stirrup
view from bottom
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Basal factors

• TATA Binding Protein (TBP)

TBP sits in the minor groove
top/side view
9

• TFIID functions

1. Recognition of core promoter 2. Target for
recruitment by transactivator proteins 3.
Binds other basal factors 4. Enzymatic activities
TAFs
TBP
mRNA
TATAAA
10

• TFIID role in core promoter recognition

TATAA binds TBP inr binds TAFs 1 2 DPE binds
TAFs 6 9
TAF6
TAF1
TAF9
TAF2
TBP
TATAAA
inr
DPE
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• TFIID role in core promoter recognition

DNA wrapping model may explain how TFIID
makes promoter contact over a large region.
TBP
TFIID
TAF1
Web site for animation of RNA polymerase binding
promoter
http//labcoulombe.usherb.ca/html/english.htm
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• TFIID TAFs serve as targets for recruitment

Acidic activation domains bind TAFs 6 9
TAF6
TAF9
TFIID
TATAAA
inr
DPE
Transactivator proteins
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TFIID TAFs contact other basal factors
5. TAF5 contacts TFIIF (RAP74)
1.TAF6 contacts TFIIE-a and TFIIF (RAP30) and
acidic activators. 2. TAF9 contacts TFIIB and
acidic activators. 3. TAF4 contacts TFIIA. 4.
TAF1 contacts TFIIF (RAP74)
TAF6
RNA pol II
E
TAF5
H
TAF1
F (RAP30)
TAF9
F (RAP74)
TAF4
TBP
B
A
(TFIIB is really on the back side of TBP)
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• TFIID Enzymatic activities of TAF1

kinase
kinase
TAND-1
BD-1
HAT
ubac
TAND-2
BD-2
Zn
Human TAFII 1
-C
1. Kinase phosphorylates TFIIF (RAP74)
significance unclear 2. Histone
acetyltransferase (HAT) adds acetyl groups
to amino tail of histones H3 and H4 loosens
chromatin structure 3. Ubiquitin
activating/conjugation activity (ubac)
monoubiquinates histone H1 significance is
unclear (?loosens chromatin?)
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• TFIID functional domains of TAF1

TAND-1
BD-1
TAND-2
BD-2
HAT
ubac
kinase
Zn
kinase
Human TAFII250
yeast TAFII145
yeast BDF-1
1
6
UbD
Arabidopsis TAFII225
3
2
7
8
2000
1000
1500
500
1
amino acids
TAND-1 and TAND-2 are N-terminal domains that
contact TBP
One copy of the largest TAF in Arabidopsis lacks
TAND-1. What may this mean in terms of regulation?
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TAND-1
BD-1
TAND-2
BD-2
kinase
HAT
ubac
Zn
kinase
TAF7 acts as a checkpoint for transcription
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The N-terminal domain of dTAF1 inhibits
transcription by binding to the concave surface
of TBP to mimic the TATAA motif. Acidic
activators are thought to also compete for
binding at the same site on TBP. It has been
proposed that acidic activators activate
transcription by displacing TAF1 binding and then
are replaced, in turn, by DNA binding to TBP.
drosTAF1 contacts TBP
TAND-1
TAND-1
BD-1
TAND-2
BD-2
HAT
ubac
kinase
Zn
kinase
-C
N-
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Dros TAF1
19
Do acidic activators compete for binding to the
concave surface of TBP?
C-
flexible hinge
TAF1
Sequential handoff mechanism?
TBP
II
TAND-I
N-terminal stirrup TBP
N-
Acidic activator
TATAAA
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TFIID has a core of histone-fold proteins
hTAF1
hTAF6

• Histone fold TAFS
• Dros human
• 62 6 80
• 42 9 31
• 30a 12 20/15
• 4b 105
• 30b 11 28
• 13 18

hTAF12
hTAF9
hTAF4b
TBP
histone-like TAFs hTAF9/hTAF6 (H3/H4) and
hTAF12 ?????/hTAF4b (H2A)
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• TFIID
• histone-like TAFs
• dros TAF9/TAF6
• (H3/H4) and
• TAF12??????/TAF4b have the potential to form an
  octamer.

TAF6
TAF9
Note the histone-fold is comprised of
three a-helices site of Dimerization.
side view
22
9
Hypothetical Structure of the nucleosome-like
TAFs in TFIID
6
Dros TFIID contains 2 TAF6 (H4) 2 TAF9 (H3) 2
TAF12 (H2b) 2 TAF4b (H2a)
Does TFIID contain a nucleosome-like structure?
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TFIID nucleosome not supported by electron
microscopy topology studies
Groove large enough to bind dsDNA. Open and
closed forms
Note TFTC does not contain TBP groove must not
be specific for presence of TBP.
Brand et al. 1999, Three-dimensional structures
of the TAFII-containing complexes TFIID TFTC,
Science 286 2151-2153.
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Three-dimensional structure of human
TFIID-IIA-IIB complex
TFIIB
Andel et al. Science 286 2153-2155.
Antibodies against TBP, TFIIB, and TFIIA used to
identify domains.
TFIIB
TFIIA
The histone fold TAFs are located in the three
lobes (A, B and C).
TBP
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Comparison of yeast and plant TFIID topology
1-TFIID has three lobes. 2- TAF5 serves as the
support scaffold for all three lobes. 3-Nucleosome
structure is not supported.
Leurent C, Sanders SL, Demeny MA, Garbett KA,
Ruhlmann C, Weil PA, Tora L, Schultz P (2004)
Mapping key functional sites within yeast TFIID.
EMBO J 23719727 _______________________ Shai J.
Lawit Æ Kevin OGrady, William B. Gurley, Eva
Czarnecka-Verner (2007) Yeast two-hybrid map of
Arabidopsis TFIID. Plant Mol Biol.
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TFIID Facts
1- TFIID is often used at promoters that lack a
TATAA motif. Note TBP can function with
complexes other than TFIID such as SAGA and
PCAF. 2- Many of the TAFs are required for
developmental processes such as gametogenesis,
sperm formation, formation of the neural tube,
and specific cell-type differentiation. 3- Many
of the TAFs can be found in other types of
transcription complexes such as SAGA and PCAF
coactivator complexes. This is especially true of
the histone-fold TAFs. Note SAGA can recruit
TBP.
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TFIID Facts
4- A number of promoters do not require TFIID for
activated transcription (those that have
TATAA). In yeast, 90 of the genes have promoters
that are primarily dependent on TFIID
(TATAA-less), a group that is largely comprised
of housekeeping genes that show basal expression
and a limited capacity for induction. The
remaining 10 have promoters that largely rely on
SAGA and are often stress inducible (including
heat inducible). 5-TBP is also involved in
transcription from RNA polymerase I and III
promoters.