Building the preinitiation complex

1
Transcriptional regulation in Eukaryotes Part
2 Genes VIII Chapters 21-22 skip sections
21.19-21.25 Genes VII Chapters 20-21
2
Arabidopsis TFIID
The yeast two-hybrid map is consistent with the
structure of yeast TFIID, suggesting conservation
of structure in eukaryotes.
Yeast 2-hybrid map of Arabidopsis TFIID, Lawit et
al. Plan Mol Biol 2007
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Yeast two-hybrid assay used to determine
protein-protein interactions
protein X
histidine
Gal4 DBD
A high resolution protein interaction map of the
yeast Mediator complex Benjamin Guglielmi, Nynke
L. van Berkum1, Benjamin Klapholz2, Theo Bijma1,
Muriel Boube2, Claire Boschiero, Henri-Marc
Bourbon2, Frank C. P. Holstege1 and Michel
Werner Nucleic Acids Research, 2004, Vol. 32,
No. 18 53795391.
Bait
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Yeast two-hybrid assay
Cloning site
Gal4 DBD
Trp
kanr
ARS
Bait vector
Selection on Leu/Trp/His dropout plates
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Arabidopsis TAF15 antisense plants
TAFII15 antisense (left 2) with wild type (right
2) Arabidopsis of the same age grown under
identical conditions. Note the small size of
rosettes in the TAFII15 antisense plants.
Closer view of TAFII15 antisense (left) with
wild type Arabidopsis plants (right). Note that
cauline leaves are the same in both plants.
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Arabidopsis TAF15 antisense plants
wild type typical leaf (1 out of 13 total)
TAFII15 antisense plant (total of 3 leaves)
NOTE cauline leaves show no differences
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RNAi (interference RNA)
Target gene
Target sequence
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Depletion of TAFII 15 mRNA by inducible RNAi
blocks seedling development
Inducible RNAi Arabidopsis Seedlings 9 Days
After Germination
- Dex
- Dex
pTA7001 empty vector control
TAFII15 RNAi T2 1
Shai Lawit unpublished
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blastocysts
TAFs play roles in development
Fig. shows expression pattern of TAF10 in mouse
embryos and adult tissues. Note the non-uniform
distribution.
Ocular muscle
Lens epithelium
tongue
Disruption (mutation) of TAF10 leads to early
embryo lethality in mice. Embryos die between
days 3.5 and 6.5.
heart
liver
TAF10 (TAFII30) Is Necessary for TFIID Stability
and Early Embryogenesis in Mice (2003) William
S. Mohan II, Elisabeth Scheer, Olivia Wendling,
Daniel Metzger, and Laszlo Tora, Mol. Cellular
Biol., June 2003, p. 43074318.
pancreas
Gut epithelium
Dorsal root ganglia
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TAFs play roles in development
heterozygous
homozygous
testes
heterozygous
homozygous
Primary spermatocytes

• TAF6 mutants in Drosophila
• Disrupts testis and sperm development.

• Eye and wing development are also affected.

Sperm tails
Developmental and Transcriptional Consequences of
Mutations in Drosophila TAFII60 NORIKAZU AOYAGI
AND DAVID A. WASSARMAN, MOLECULAR AND CELLULAR
BIOLOGY, 2168086819.2001.
Egg shells
wt
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Basal factors

• Other complexes also
• contain TAFS

SAGA complex
TFIID
SPTs
(yeast)
(histone-fold)
TBP
TBP
TAF Subset (histone-fold)
GCN4
ADA/ GCN5 (histone acetyltransferase)
TATAA
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Alternate complexes containing TBP/TAFs
human
yeast
SAGA
PCAF
13
Basal factors
TFIIA 1. binds to N-terminal stirrup of TBP. 2.
displaces negative factors which also bind TBP
(NC1 NC2/DR1). 3. stabilizes TBP/DNA.
TAF6
TFIIE
RNA pol II
(12 subunits)
F
TAF9
TFIIF
TAF4
TBP
B
TFIIA
TAF150
TFIIB
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Basal factors

• TFIIA/TBP/DNA complex

1
TFIIA (2 subunits)
TFIIA attaches to the N-terminal stirrup of TBP
and stabilizes the TBP/DNA complex
TBP
view from bottom
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Basal factors

• TFIIA/TBP/DNA complex

TFIIA contacts TBP N-stirrup
top view
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TFIIB 1. binds to C-terminal stirrup of TBP. 2.
Binds TFIIF (RAP30) which in turn is bound to RNA
pol II. 3. stabilizes TBP/DNA. 4. target for
activator proteins.
Basal factors
TFIIE
TAF6
RNA pol II
(12 subunits)
F
TAF9
TFIIF
TAF4
TBP
B
TFIIA
TAF2
TFIIB
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Transcription factor IIB
basic amphipathic helix




B finger
linker
-C
N-
TFIIB
Zn ribbon
R1
R2
Linker 17 aa human, rat, frog, fly 23aa
plants 29aa yeast.
regions of VP16 interaction
4
Structure of conserved core (R1 R2)
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Bagby et al., 1995, Cell 82 857-867
Linker
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Building the preinitiation complex (PIC)
TFIIA and TFIIB bind to TBP and stabilize complex
with DNA 1. TFIIA binds to N-terminal stirrup of
TBP. 2. TFIIB binds to C-terminal stirrup of TBP.
TBP
TATAA
TFIIA
TFIIB
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Basal factors

• TFIIB/TBP/DNA complex

RAP30 binds here
TFIIB
TFIIB contacts C-stirrup of TBP
N-
C-
1
TBP
top view
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Structural basis of transcription an RNA
polymerase II-TFIIB cocrystal at 4.5 Angstroms,
Bushnell, Westover and Kornberg (2004) Science
303983-988.
C-terminal core domains
-N
Two highly conserved domains at the N-terminus of
TFIID make contact with RNA pol II and help
position the template strand.
21
The Zn ribbon and B finger are positioned near
the active site and within the RNA exit pore of
RNA pol II.
Structural basis of transcription an RNA
polymerase II-TFIIB cocrystal at 4.5 Angstroms,
Bushnell, Westover and Kornberg (2004) Science
303983-988.
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The B finger stabilizes the initial short
transcript within the RNA exit pore.
Structural basis of transcription an RNA
polymerase II-TFIIB cocrystal at 4.5 Angstroms,
Bushnell, Westover and Kornberg (2004) Science
303983-988.
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Domains and structure of TFIIB
-N
R1
R2
Mapping the Location of TFIIB within the RNA
Polymerase II Transcription Preinitiation Complex
A Model for the Structure of the PIC, (2004)
Chen, H.-T. Hahn, S., Cell 119169-180.
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TBP
Model for the location of TBP, TFIIB and RNA pol
II in the preinitiation complex (PIC)
R1
R2
Zn ribbon
Mapping the Location of TFIIB within the RNA
Polymerase II Transcription Preinitiation Complex
A Model for the Structure of the PIC, (2004)
Chen, H.-T. Hahn, S., Cell 119169-180.