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Molecular and Cellular Biology, March 2001, p. 1737-1746, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1737-1746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
TFIIA Interacts with TFIID via Association with
TATA-Binding Protein and TAF40
Susan M.
Kraemer,1
Ryan T.
Ranallo,2
Ryan C.
Ogg,1 and
Laurie A.
Stargell1,*
Department of Biochemistry and Molecular
Biology, Colorado State University, Fort Collins, Colorado
80523-1870,1 and Laboratory of Molecular
Cell Biology, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892-42552
Received 30 August 2000/Returned for modification 29 October
2000/Accepted 22 November 2000
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ABSTRACT |
TFIIA and TATA-binding protein (TBP) associate directly at the TATA
element of genes transcribed by RNA polymerase II. In vivo, TBP is
complexed with approximately 14 TBP-associated factors (TAFs) to form
the general transcription factor TFIID. How TFIIA and TFIID communicate
is not well understood. We show that in addition to making direct
contacts with TBP, yeast TAF40 interacts directly and specifically with
TFIIA. Mutational analyses of the Toa2 subunit of TFIIA indicate that
loss of functional interaction between TFIIA and TAF40 results in
conditional growth phenotypes and defects in transcription. These
results demonstrate that the TFIIA-TAF40 interaction is important in
vivo and indicate a functional role for TAF40 as a bridging factor
between TFIIA and TFIID.
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INTRODUCTION |
Transcription by eukaryotic RNA
polymerase II (Pol II) involves the assembly of a preinitiation complex
consisting of Pol II and the general transcription factors TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (for review, see reference
66). An important step of transcription initiation is the
binding of TFIID to the core promoter. TFIID, a multisubunit protein
complex that is highly conserved among eukaryotes, is composed of the
TATA-binding protein (TBP) and over a dozen TBP-associated factors
(TAFs) (reviewed in references 26 and 27). TBP mediates
promoter recognition through the sequence-specific binding of the TATA
element found at many promoters. The importance of the TBP-TATA
interaction is illustrated by many studies which demonstrate that
recruitment of TBP is a rate-limiting step at a majority of promoters
(12, 17, 37, 39, 46, 50).
In yeast, 13 TAFs are required for viability, indicating essential
roles for individual TAFs (75, 77; reviewed in reference 26). However, the precise functional requirements for the
TAFs remain unresolved. In vitro biochemical experiments suggest that TAFs function in higher eukaryotic systems as obligatory coactivators essential for activator response (reviewed in references 9 and
84). In contrast, functional inactivation and depletion studies
with certain TAFs in yeast cells demonstrate that the expression of
many genes are unaffected by TAF loss, although TAF inactivation
results in distinct cell cycle phenotypes (2, 55, 59, 60, 62,
86). In addition, disruption of the TFIID complex with a
temperature-sensitive mutation in TBP results in gene-specific
transcriptional defects (71), and promoter occupancy
studies indicate that TAFs are not present on certain transcriptionally
active promoters in vivo (45, 49). Promoter-specific requirements for particular TAFs are further illustrated by
whole-genome transcriptional profiles. For example, inactivation of
TAF145/130 has no effect on the expression of a majority of genes,
while transcription of a subset of genes is affected (31).
This TAF dependence was mapped to the core promoter (78),
indicating important TAF functions in promoter activity in vivo. In
contrast to these gene-specific effects, inactivation of several other TAFs, namely TAF17 (2, 59, 61), TAF40 (44),
TAF60 and TAF61/68 (59, 62), and TAF23/25
(76), results in dramatic effects on a large fraction of
genes transcribed by Pol II. The requirement for these particular TAFs
is not yet understood, but it is clear that individual TAFs may be
generally required for transcription while others function at a subset
of promoters. To complicate the issue further, it is apparent that
certain TAFs in both human and yeast systems can be found in large
protein complexes distinct from TFIID, such as the SAGA complex
(25) and the SWI/SNF complex (10). Taken
together, these studies indicate that different TAFs may have distinct
functional roles in transcription, yet the nature of the specific
functions for a majority of the TAFs remains to be elucidated.
In addition to TAFs, other transcription factors associate with TBP at
the core promoter to mediate transcription. One such factor, TFIIA, has
been shown to stabilize the interaction between TBP and DNA at the TATA
element (reviewed in reference 27). Mutational studies of
both TBP and TFIIA demonstrate the importance of the TFIIA-TBP
interaction for transcription in vivo (35, 53, 68, 80).
TFIIA has been described as a coactivator, since in vitro functions of
certain activators are TFIIA dependent (51, 67), and as an
antirepressor, because TFIIA can mediate displacement of certain
transcriptional inhibitors that act on TBP (4, 5, 22, 32, 42, 52,
56, 58, 69).
A growing body of evidence suggests that the functions of TFIIA and
TAFs are connected. DNase I footprinting experiments show that the
addition of TFIIA alters the DNA protection pattern of TFIID (14,
15, 51). Consistent with these findings, UV-cross-linking experiments indicate that TFIIA induces a conformational change in
TFIID that alters specific TAF interactions with the core promoter (64). Moreover, a set of TFIIA mutations that can form a
TBP-TFIIA-DNA complex are defective for forming a complex with TFIID
(69). Finally, the three-dimensional structure of the
human TFIID-TFIIA-TFIIB complex clearly suggests TFIIA-TAF
interactions, since TFIIA maps to a large noncentral lobe of TFIID,
with TBP being located more centrally in the structure
(1). It is not yet understood how this TFIIA-TAF
communication is established or which particular TAFs are involved.
In this report, we investigate the importance of the interactions
between TFIIA and yeast TAFs. We demonstrate a direct interaction between TFIIA and TAF40, as well as a direct interaction between TAF40
and TBP. We also find that mutations in TFIIA that impair the
TFIIA-TAF40 interaction result in conditional growth phenotypes and
defects in transcription in vivo. These results suggest that TAF40
serves as a link between TFIIA and TFIID functions, and they reveal a
new role for TAF40 in RNA Pol II transcriptional regulation.
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MATERIALS AND METHODS |
DNA constructs.
Activation domain (AD) hybrids were cloned
into the 2µ LEU2 marked vector, pACT2.2 (19),
which contains the ADH1 promoter, a nuclear localization
sequence, the hemagglutinin (HA) epitope, and the Gal4 AD (residues 768 to 881). DNA-binding domain (DB) hybrids were created by subcloning
from the corresponding AD constructs into the pPC97-TRP
vector (85) (CEN, TRP3), which contains the ADH1 promoter, a nuclear localization sequence, and the Gal4
DB (residues 1 to 147). The TAF40 Escherichia coli
expression plasmid was created by cloning the TAF40 open reading frame
into the pET15b vector using PCR and designed oligonucleotides. The
E. coli expression plasmids for Toa2 glutathione
S-transferase (GST)-TFIIB have been described elsewhere
(80). GST-Toa1 was constructed by subcloning an
EcoRI fragment containing the open reading frame of Toa1
into the pGEX1
T vector (Pharmacia); GST-TBP was constructed by PCR of TBP followed by cloning into the GST-2T vector (Pharmacia). TOA2-YCP22 contains TOA2 driven by its native promoter and
terminator, which were generated by PCR from genomic DNA. A
NcoI site was engineered at the ATG start codon and utilized
for inserting six myc epitopes (GEQKLISEEDLN), creating myc-TOA2-YCP22.
Site-directed TOA2 mutants were created using
oligonucleotide primers containing the desired mutation and PCR. Mutant
derivatives were subsequently subcloned into the Gal4-DNA binding
domain vector (pPC97-TRP1) and into pET15b (Novagen) lacking
the histidine tag. All PCR products were completely sequenced.
Yeast strains.
All strains used in the yeast two-hybrid
assay were transformants of MaV103 (85). MaV103 contains
the GAL1 promoter (with four Gal4 binding sites) fused to
the HIS3 promoter and structural gene; GAL4 and
GAL80 are both deleted in the strain. Viability tests of
TOA2 mutant derivatives were conducted in ROY100, a
derivative of KY114 (relevant genotype MATa
ade2-101 leu2::PET56 trp1
1 ura3-52), which was
created using a two-step gene knockout of the complete open reading
frame of the TOA2 gene and contains TOA2 on a 2 µm, URA3-marked plasmid. The plasmid shuffle technique, which involves transforming ROY100 with TRP1-marked TOA2
derivatives, followed by selection for loss of the
URA3-marked plasmid by growth on 5-fluoro-orotic acid
(5-FOA), was used to test the mutant derivatives for viability and to
create Toa2 mutant strains for further characterization.
Yeast two-hybrid assays and phenotypic studies.
Both Gal4 DB
and Gal4 AD plasmids were transformed into the yeast strain MaV103
using a standard lithium acetate transformation. The resulting strains
were grown in the appropriate selection media, and 10-fold serial
dilutions were performed. Cells were spotted onto the appropriate
plates, which either contained or lacked 3-aminotriazole (AT), and
grown at 30°C for 4 to 7 days. For phenotypic studies, 10-fold serial
dilutions of strains were spotted on plates with rich media containing
either glucose (yeast-peptone-dextrose [YPD]) or galactose
(yeast-peptone-galactose [YPG]), and plates were incubated at either
30 or 38°C.
Protein purification.
TFIIA was purified as described
previously (74) by expressing the GST-Toa1 and -Toa2
subunits separately in E. coli, denaturing both in 8 M urea,
combining the subunits, and dialyzing out the urea. GST-TBP, GST-TFIIB,
and GST were expressed and purified from bacteria as described
elsewhere (80). TAF40 was purified with a denaturing and
refolding method similar to that used to make recombinant yeast TFIIA.
BL21 (DE3) cells containing TAF40 cloned into the bacterial expression
plasmid pET15b (His-TAF40) were grown to an optical density at 600 nm
(OD600) of 0.6 and induced for 2 h with 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG). One liter of
cells was harvested, washed with 200 ml of buffer A (20 mM Tris-HCl
[pH 7.5], 200 mM NaCl), and resuspended in 25 ml of buffer 1 (20 mM
Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol
[DTT], 0.05% NP-40), and frozen at 
70°C. Cells were thawed and
sonicated. The insoluble fraction was collected by spinning at
10,000 × g for 15 min at 4°C. Pellets were
resuspended in 30 ml of buffer 2 (20 mM Tris-HCl, 50 mM NaCl, 1 mM
EDTA, 8 M urea, 5 mM DTT). A 7.5-ml volume of buffer 3 (20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM DTT, 10% glycerol) was added slowly, and
the solution was cleared by spinning at 10,000 × g for 15 min at 4°C. The supernatant was dialyzed against buffer 3 containing 1 mM imidazole. The dialyzed proteins were cleared by spinning at
10,000 × g for 15 min at 4°C. The soluble material
was bound to Ni-nitrilotriacetic acid resin (Qiagen), washed with wash
buffer (20 mM Tris [pH 7.5], 100 mM KCl, 10% glycerol, 40 mM
imidazole, 1 mM DTT), and eluted with elution buffer (20 mM Tris-HCl
[pH 7.5], 100 mM KCl, 10% glycerol, 1 mM DTT, 200 mM imidazole).
Elution fractions were dialyzed against buffer containing 20 mM HEPES (pH 7.9), 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10% glycerol, and 1 mM DTT.
In vitro interaction studies.
Approximately 12.5 pmol of
GST-fusion protein or GST alone was incubated with 10 to 20 pmol of
His-TAF40 protein in 200 µl of binding buffer (20 mM HEPES [pH
7.9], 20 mM Tris [pH 7.5], 200 mM NaCl, 50 mM KCl, 10 mM
MgCl2, 0.025% NP-40, 10% glycerol, 0.5 mM DTT) for 3 h at 4°C. Complexes were recovered by incubation with glutathione
Sepharose for 1 h at 4°C in binding buffer with 3% bovine serum
albumin. Complexes were washed two times in 400 µl of binding buffer,
incubated with sodium dodecyl sulfate (SDS) loading buffer, and boiled,
and 10 µl of sample was separated by SDS-polyacrylamide gel
electrophoresis (PAGE). Gels were analyzed by immunoblotting with
antibodies specific to His-TAF40 (Santa Cruz Biotechnology) or GST
(Sigma) and visualized by chemiluminescence detection (Pierce).
Electrophoretic mobility shift assays were performed using a
32P-labeled 45-bp fragment containing the adenovirus early
1B TATA box, as described previously (80). Purified
recombinant yeast TBP (5 nM), TFIIA or TFIIB (3 nM), TAF40 (19 to 142 nM), and 100 ng of poly(dG-dC) were incubated at 25°C for 30 min in
20 µl of 20 mM Tris (pH 7.5), 40 mM HEPES (pH 7.9), 100 mM KCl, 1 mM
DTT, 0.5 mM phenylmethyl sulfonyl fluoride, and 10% glycerol.
Complexes were separated from unbound DNA by 6% nondenaturing
acrylamide gel electophoresis in 0.5× Tris-borate-EDTA and quantified
by phosphorimaging.
Transcriptional analysis.
Quantitative S1 nuclease analysis
was done as described elsewhere (34), with approximately
30 to 50 µg of RNA. For the temperature shift and AT (Sigma)
inductions, cells were grown in synthetic complete medium to an
OD600 of 0.5 to 1.0. Cells were pre-heat shocked at 38°C
for 15 min and incubated at 30°C for 1 h, followed by 38°C for
1 h. AT was added to a concentration of 20 mM, and cells were incubated
for an additional hour at 38°C. Total RNA was prepared by hot-phenol
extraction and was quantitated at OD260. RNA amounts in
each reaction mixture were normalized to the levels obtained from a
probe to the intron of the tryptophan tRNA gene (tRNAW).
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RESULTS |
Yeast TAF40 associates with TFIIA in vivo.
TFIIA
interacts with TBP and DNA at the promoter and stabilizes the TBP-DNA
interaction. Yet, within a cell, TBP associates with TAFs to form the
TFIID complex. We used a yeast two-hybrid assay to investigate the
potential interplay between TFIIA and TAFs. Yeast TFIIA is composed of
two subunits encoded by the genes TOA1 and TOA2,
both of which are required for viability (73). The DB of
Ga14 was fused in frame with Toa2 (DB-Toa2), creating the bait for the
two-hybrid assay. TAF proteins were fused in frame to the Ga14 AD.
Fusion proteins were expressed in a yeast strain with the
HIS3 gene under the control of the GAL1 promoter (which contains four Ga14 binding sites). Interactions between Toa2 and
the AD-fusion proteins were determined by examining activation of the
HIS3 gene. HIS3 gene activation was assayed by
growth in the presence of AT, a competitive inhibitor of the
HIS3 gene product (28). Strains in which the
HIS3 gene is highly expressed, due to interactions between
the DB-fusion protein and the AD-fusion protein, will grow on AT.
In contrast to a DB fusion to Toa1, which activates transcription
(
79), expression of DB-Toa2 showed no
HIS3 gene
activation
(Fig.
1A). The difference
between the activity of the two subunits
is not understood, but it does
provide the opportunity to use
DB-Toa2 in the two-hybrid assay. Since
DB-Toa2 exhibited a strong
interaction with AD-Toa1, DB-Toa2 is not
defective for subunit
interactions with Toa1, and this also suggests
that DB-Toa2 interacts
with native Toa1 within the cell. We then tested
the ability of
DB-Toa2 to interact with all 13 of the known essential
yeast TAFs
found in the TFIID complex (for review, see references
26 and
27). A strong interaction was observed between Toa2
and TAF40.
Because two-hybrid interactions can be indirect and TAF40 is
a
component of the TFIID complex, it was surprising that DB-Toa2
showed
no interaction with the other TAF proteins shown in Fig.
1 (TAF17,
TAF19, TAF47, and TAF61) or TAF25, TAF48, TAF60, TAF65,
TAF67, TAF90,
TAF130, and TAF150/TSM1 (data not shown). The lack
of interaction was
not due to lack of expression, since each of
the TAF fusion proteins
was easily detectable by immunoblotting
of whole-cell extracts with
antibodies specific to the HA tag
present in the AD vector (data not
shown). These results suggest
that TAF40 associates with Toa2 within
the cell, perhaps directly.
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FIG. 1.
TFIIA associates with TAF40 in vivo. (A) Two-hybrid
assays were used to demonstrate that TFIIA interacts specifically with
Toa1 and TAF40. The indicated Gal4 AD fusion proteins were tested for
the ability to interact with a DB fusion of Toa2 (DB-Toa2).
Approximately 104 cells were spotted onto synthetic
complete plates containing either 0 or 40 mM AT. Growth on AT is
indicative of an interaction between the two hybrid proteins. The
bottom panel shows that strains containing AD-Toa1 or AD-TAF40 do not
grow on AT with the DB vector alone. (B) TAF40 stimulates transcription
in an artificial recruitment assay. DB-TBP, DB-40, and DB-Fos each
stimulate high levels of transcription when tethered to a promoter via
a heterologous DB.
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A reciprocal interaction between TAF40 and TFIIA could not be examined
because expression of the fusion of TAF40 to the DB
resulted in high
levels of transcriptional stimulation (Fig.
1B).
This indicates that
recruitment of TAF40 to a promoter is sufficient
to stimulate
transcription of that gene. This result is in accord
with similar
recruitment studies that used other TAFs and TBP
(
3,
7,
12,
21,
24,
36,
37,
89).
TFIIA has been shown to interact with certain ADs (
16,
40,
70). A trivial explanation for the interaction detected is
that
DB-Toa2 interacts with the AD of AD-TAF40, and the TAF40
domain
stimulates transcription. To examine this possibility,
the Fos
C-terminal AD, which strongly stimulates transcription
when bound to
the promoter via the Ga14 DB (Fig.
1B), was cloned
into the Ga14 AD
vector (AD-Fos). Expression of the AD-Fos hybrid
protein, which has two
tandem functional ADs, did not yield a
positive two-hybrid interaction
with DB-Toa2 (Fig.
1A). The inability
of DB-Toa2 to interact with
AD-Fos eliminates the possibility
that Toa2 is simply interacting with
the Ga14 AD of AD-TAF40 and,
in effect, mimicking the activation by
DB-TAF40 seen in the artificial
recruitment
assay.
TAF40 interacts with TFIIA and TBP.
To demonstrate a direct
interaction between TAF40 and TFIIA or TBP, recombinant proteins were
produced in bacteria and their ability to physically interact was
examined using GST pull-down assays. Immunoblot analysis of the
isolated complexes revealed that significant amounts of TAF40 interact
with the GST-TFIIA (Fig. 2A). In
addition, GST-TBP interacts with TAF40 directly. TAF40 does not
interact with either GST-TFIIB or GST.
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FIG. 2.
TAF40 interacts directly with TFIIA or TBP. (A) To test
whether TFIIA interacts directly with TAF40 in vitro, a GST
pull-down assay was used. Recombinant GST-TFIIA, GST-TBP,
GST-TFIIB, or GST was incubated with recombinant histidine-tagged TAF40
(TAF40). After SDS-PAGE, TAF40 was detected using antibodies
specific to the histidine tag of TAF40. Antibodies specific to the GST
tag were used to confirm that similar amounts of GST and the GST fusion
proteins were utilized (data not shown). (B) TAF40 increases
TBP-TFIIA-DNA complex formation on a TATA box. For all reactions,
9 nM radiolabeled adenovirus early 1B TATA box probe was used.
Concentrations of TBP and TFIIA were held constant at 5 and 1.5 nM,
respectively. Lane 1 contains TBP, lane 2 contains TBP and TAF40 (142 nM), lane 3 contains TFIIA, lane 4 contains TFIIA and TAF40 (142 nM).
Lane 5 contains TAF40 (142 nM) alone. Lane 6 contains TBP and TFIIA,
and lanes 7 through 11 contain TBP and TFIIA with increasing amounts of
TAF40 (19 to 142 nM). (C) TAF40 does not increase the TBP-TFIIB complex
formation on a TATA box. All reactions are as in panel B, except that 3 nM TFIIB is used in place of TFIIA.
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Electrophoretic mobility gel shift assays were used to test whether the
presence of TAF40 affects the TBP-TFIIA-DNA ternary
complex. In the
absence of magnesium, TBP does not form a stable
complex with DNA, and
the addition of TFIIA stabilizes the TBP-DNA
interaction and shifts the
TATA-containing probe (Fig.
2B). Under
conditions of subsaturating
amounts of TFIIA, the addition of
TAF40 resulted in a 20-fold
enhancement of the TFIIA-TBP-DNA complex
(Fig.
2B). Enhancement of the
TBP-TFIIA-DNA complex was not observed
when equivalent amounts of
bovine serum albumin or buffer were
added (data not shown). In
addition, TAF40 had no effect on the
amount of ternary complex formed
with TFIIB (Fig.
2C). Excess
TAF40 also resulted in no changes in the
DNA-binding properties
of TBP, TFIIA, or TFIIB alone, either in the
presence or absence
of magnesium ions (Fig.
2B and C and data not
shown). Thus, enhancement
of complex formation by TAF40 is specific to
the TBP-TFIIA-DNA
ternary
complex.
Although TAF40 addition results in a significant enhancement of the
TBP-TFIIA-DNA complex, it does not alter the mobility
of the complex.
This result suggests that either the addition
of TAF40 to the complex
results in a conformational change that
masks the added mass of TAF40,
or that TAF40 is not stable to
the gel running conditions. We feel that
the latter hypothesis
is supported by the observation that inclusion of
antibodies specific
to TAF40 does not result in a shift in the complex
size but instead
abolishes the ability of TAF40 to enhance
TBP-TFIIA-DNA complex
formation (data not
shown).
Mutations in a hydrophobic patch of TFIIA are defective for
interacting with TAF40 in vivo.
Analysis of the crystal structures
of the yeast TFIIA-TBP-DNA ternary complex (23, 81)
reveals several striking features. TFIIA consists of two domains, a domain and a four-helix bundle (4HB) domain (Fig.
3). The domain makes all of the
contacts with TBP and also binds DNA upstream of the TATA element. The 4HB domain of TFIIA projects away from the TBP-TFIIA-DNA complex into
solution. In addition, there are two large solvent-exposed patches of
hydrophobic residues on TFIIA; one patch is within the domain and
the other is within the 4HB domain. Hydrophobic interactions are
important for many protein-protein interactions. In fact, the
hydrophobic region on the domain contacts TBP. We hypothesized that
the hydrophobic patch on the 4HB domain may contribute to other TFIIA
functions, possibly for interactions with TAF40. Three residues within
this hydrophobic patch were targeted for mutational studies: isoleucine
at position 27 of Toa2 was changed to alanine (I27A) or lysine (I27K);
methionine at position 38 was changed to alanine (M38A) or lysine
(M38K); and leucine at position 41 was substituted with either alanine (L41A) or aspartic acid (L41D).
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FIG. 3.
Crystal structure of the TFIIA-TBP-DNA complex showing
the location of the amino acids replaced in the hydrophobic region of
the 4HB. TBP is shown in a yellow ribbon, and DNA is black. TFIIA is
shown in the space-filling model, with Toa1 in dark blue and Toa2 in
light blue. The two hydrophobic patches on TFIIA are shown in gray; one
patch contacts TBP. The amino acids in Toa2 selected for replacement by
alanine or radical amino acids are shown in magenta and are indicated
by the arrows and the labels. The figure was created with Insight II,
using the coordinates of the TBP-TFIIA-DNA structure
(81).
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Each of the mutant derivatives was cloned into the DB vector. To
determine whether any of the mutations causes a drastic change
in the
folding ability of Toa2, the derivatives were tested for
the ability to
interact with AD-Toa1 (Fig.
4). DB-L41D
was defective
for interacting with Toa1, indicating that this Toa2
derivative
is compromised for TFIIA formation. The five remaining
derivatives
were indistinguishable from wild-type Toa2 with regard to
their
ability to interact with Toa1.
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FIG. 4.
Mutations in Toa2 impair interactions with TAF40. The
two-hybrid assay was used to identify mutations in the hydrophobic
patch of the TFIIA 4HB that are defective for interaction with TAF40.
The AD constructs are indicated across the top, and the DB-Toa2
derivatives are shown along the left side. Approximately
104 cells were spotted onto plates containing 40 mM AT. All
strains grew robustly on media lacking AT (data not shown).
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We next tested the Toa2 derivatives for defects in interactions with
TAF40. I27A, I27K, and L41D showed significantly weakened
interactions
with TAF40 in the two-hybrid assay (Fig.
4). Since
L41D was also
defective for interaction with Toa1, the loss of
the TAF40 interaction
may be the result of global defects in the
structure of this protein.
In contrast, substitutions at I27 are
specifically defective for TAF40
interactions, in that both I27A
and I27K are indistinguishable from
wild-type Toa2 for Toa1 interactions.
The remaining derivatives
exhibited interactions with both TAF40
and Toa1 that were comparable to
that with wild-type Toa2. These
results suggest that the hydrophobic
region on the 4HB domain
of TFIIA, in particular residue I27 of Toa2,
plays an important
role in the interaction between TFIIA and
TAF40.
Toa2 derivatives impart mutant growth phenotypes.
To examine
the physiological relevance of the TFIIA-TAF40 interaction, the I27A
and I27K Toa2 mutants (under the control of the TOA2
promoter and terminator) were expressed in a TOA2 deletion strain. Both alleles supported cell viability, but each caused a
slow-growth phenotype at 30°C and a temperature-sensitive phenotype at 38°C (Fig. 5A). The doubling time
for wild-type cells was 2.5 h, whereas I27A- and I27K-containing
cells have a doubling time of 3.5 to 4 h at 30°C (data not
shown). The slow-growth phenotype at 30°C is consistent with the
observation that the TFIIA-TAF40 interaction is disrupted in the
two-hybrid assay, which is performed at this same temperature (30°C).
Furthermore, the I27K mutant was unable to support growth on
galactose-containing medium, suggesting an inability to respond to the
Gal4 activator protein. Mutant phenotypes were not the result of a
destabilization of Toa2 protein, since the I27A and I27K strains
produced amounts of Toa2 protein comparable to that in wild-type Toa2
at 30 and 38°C, as assayed by immunoblot analyses of whole-cell yeast
extracts (Fig. 5B). None of the mutant phenotypes could be suppressed
by overexpression of TAF40 (data not shown). This suggests that
interactions with TAF40 and the I27A and I27K forms of TFIIA are
severely compromised, since simple overexpression is not sufficient to
counteract the loss of interaction.
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FIG. 5.
TAF40-defective Toa2 derivatives confer mutant
phenotypes. (A) Strains containing the I27A and I27K mutations of Toa2
were tested for conditional phenotypes in a TOA2 deletion
strain. The indicated strains were serially diluted (from
104 to 10 cells) and spotted onto rich media plates
containing glucose (YPD) or galactose (YPG) and incubated at either 15, 30, or 38°C. (B) Levels of Toa2, I27A, and I27K proteins are
indistinguishable. Strains containing wild-type Toa2 in addition to
either myc-tagged wild-type Toa2 (WT), myc-tagged I27A (I27A), or
myc-tagged I27K (I27K) were harvested after incubation at 30 or 38°C.
Extracts (20 µg) were subjected to SDS-PAGE and immunoblotting with
anti-myc antibodies and anti-TBP antibodies (for a load control). The
presence of wild-type Toa2 in each of the strains allows for an
accurate analysis of the stability of the mutant derivatives, since
cell viability is not dependent on their expression.
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In vitro analysis of the TFIIA mutants I27A and I27K.
In order
to determine the extent to which the mutations at the I27 residue were
defective for the interaction with TAF40 in vitro, GST pull-down assays
were performed. Immunoblot analysis of the recovered complexes showed
that TFIIA recombinant proteins containing the I27A and I27K
substitutions were significantly decreased in their ability to interact
with TAF40 when compared to wild-type TFIIA (Fig.
6A). Moreover, TAF40 binding by I27K was
indistinguishable from the binding observed with GST alone. These
results coincide with those of the two-hybrid studies, since in both
the two-hybrid assay and the pull-down assay, I27K displayed a more
dramatic defect in interacting with TAF40. In contrast, TFIIA
substituted with I27A or I27K is fully functional in forming the
TBP-TFIIA-DNA complex (Fig. 6B). In addition, both I27A- and I27K-substituted TFIIA were competent for complex enhancement by TAF40
(Fig. 6B). This result is not unexpected, since the direct interaction
between TBP and TAF40 (Fig. 2) may compensate for the loss of
interaction between TAF40 and TFIIA.
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|
FIG. 6.
: In vitro analysis of the TFIIA mutants I27A and I27K.
(A) To test the ability of TFIIA substituted with I27A and I27K to
interact with TAF40, a GST pull-down assay was used. Recombinant GST,
GST-TFIIA (WT), and TFIIA formed with the I27A (GST-I27A) and I27K
(GST-I27K) mutants were incubated with recombinant histidine-tagged
TAF40. The complexes were isolated by incubation with GST resin
followed by washing. Samples were separated by SDS-PAGE and analyzed by
immunoblotting using antibodies specific to the histidine tag of TAF40.
Immunoblot analysis was also done using antibodies specific to GST to
assay the amounts of GST and GST-fusion proteins recovered. (B) TFIIA
substituted with either I27A or I27K is fully functional in forming the
TBP-TFIIA-DNA complex, and formation of the complex is enhanced by
TAF40. For all reactions, 9 nM radiolabeled adenovirus early 1B TATA
box probe was used. Lanes contain TBP (5 nM), WT or mutant TFIIA (1.5 nM), or TAF40 (142 nM) as indicated.
|
|
TFIIA mutants are defective for transcription in vivo.
To
determine the ramifications of a defect in the TFIIA-TAF40 interaction,
we compared wild-type, I27A, and I27K strains for their transcriptional
competency. Constitutive transcription of a collection of Pol
II-transcribed genes was examined at 30 and 38°C (Fig.
7). When cultured at 30°C, the I27A and
I27K strains exhibited a significant reduction in HIS3 gene
expression compared to expression in wild-type cells. Levels of
transcription of both the +1 and +13 transcripts of HIS3
were decreased. The +1 transcript is generated from a noncanonical
promoter element, while the +13 transcribed is derived from a
conventional TATA element (13, 33). This suggests that the
TAF40-TFIIA interaction is important for transcription from both
canonical and noncanonical promoters in vivo. RPS4, DED1,
HTA2, and PGK1 mRNA levels also decreased at both 30 and 38°C. The decreases in transcription at the permissive temperature (30°C) are consistent with the fact that the TAF40-TFIIA interaction defect is observed at 30°C in the two-hybrid assay. The
transcriptional effects on expression of RPS4,
HTA2, and DED1 in strains with defective
TFIIA-TAF40 interactions are consistent with similar observations on
the expression of these genes in a TAF40 mutant strain
(44). In contrast to the genes described above,
transcription from the ENO2 gene was unaffected. Thus, requirement for the TFIIA-TAF40 interaction is promoter specific. Promoter-specific dependency on TAF40 is also supported by chromatin immunoprecipitation studies, which showed TAF40 occupancy can vary on
transcriptionally active promoters (45).
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FIG. 7.
Transcriptional analysis of the TFIIA mutants defective
in the TAF40 interaction for a collection of Pol II-transcribed genes.
Wild-type (WT) and mutant Toa2 (I27A or I27K) strains were grown to log
phase and shifted to the restrictive temperature for 1 h. Thirty to 50 µg of total RNA was hybridized with a 100-fold excess of the
indicated probe and treated with S1 nuclease. The HIS3 +1
and +13 initiation sites are indicated. The Pol III-transcribed gene
tRNAw served as a loading control.
|
|
We also tested the ability of the I27A and I27K strains to respond to
acidic activators. Gcn4-dependent activation of
HIS3 transcription was assayed by growing the cells in AT, a competitive
inhibitor of the
HIS3 gene product (Fig.
8A). To examine the response
at the
restrictive temperature, cells were incubated at 38°C for
1 h,
AT was added, and the cells were incubated for an additional
hour
before harvesting. For both the I27A and I27K mutants, activation
of
HIS3 transcription was decreased compared to that in
wild-type
cells. Activation by the acidic activator Gal4 was determined
by growing cells in galactose-containing medium and assaying for
GAL1 transcript levels (Fig.
8B). Both the I27A and I27K
mutants
displayed a significant decrease in
GAL1
transcription. Taken
together, these defects in both constitutive and
induced transcription
for the I27A and I27K derivatives of Toa2
indicate that the TAF40-TFIIA
interaction plays an important role in
transcription in vivo.
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FIG. 8.
Transcriptional analysis of activator-induced genes. (A)
Analysis of Gcn4-dependent activation of HIS3 transcription.
For assays at 30°C, 20 mM AT was added for 1 h, followed by
harvesting of the cells. For assays at 38°C, cells were grown to
early log phase, shifted to 38°C for 1 h, and then grown for an
additional hour in the presence of 20 mM AT. Total RNA was analyzed for
HIS3 and tRNAw expression by S1
analysis. (B) Analysis of Gal4-dependent activation of GAL1
transcription. Strains were grown in raffinose-containing medium to
early log phase and then grown for the indicated time in galactose at
30°C. Total RNA (30 to 50 µg) was hybridized with 100-fold excesses
of GAL1 and tRNAw probes and then
subjected to S1 nuclease digestion.
|
|
TFIIA mutants defective for interactions with both TAF40 and TBP
are not viable.
Combining previous work and the results presented
above, we conclude that TFIIA interacts with TFIID via TAF40 and TBP.
To determine how critical the contacts with TAF40 and TBP are for TFIIA
functions in vivo, mutations defective for the TFIIA-TAF40 interaction
and the TFIIA-TBP interaction were engineered into the same Toa2
allele. TFIIA substituted with a Y69A mutation in Toa2 is not competent
for forming the TBP-TFIIA complex in vitro and results in a
temperature-sensitive phenotype and transcriptional defects in vivo
(68). We tested the hypothesis that TFIIA interactions with TFIID are essential by combining mutations in Toa2 at either I27A
or I27K (which are TAF40 defective) with Y69A (which is TBP defective).
While the I27A, I27K, and Y69A single mutants were able to support cell
viability, both the I27A/Y69A and I27K/Y69A double mutants were unable
to support cell viability in a strain deleted for TOA2 (Fig.
9). Thus, TFIIA interactions with TFIID via TBP or TAF40 are essential for cell survival.
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FIG. 9.
TFIIA mutants defective for TAF40 and TBP interaction
cannot support cell viability. The growth of strains containing the
indicated Toa2 derivative (or vector) was analyzed by spotting
approximately 104 cells onto plates deficient for
tryptophan and containing (+) or lacking () 5-FOA. When wild-type
Toa2 (carried on a URA3 plasmid) is shuffled out on 5-FOA,
cell viability is dependent on Toa2 functions carried on the
TRP1 plasmid. WT, wild type.
|
|
| |
DISCUSSION |
TFIIA and TAF40 interact directly.
The ability of TFIIA to
interact with TBP and stabilize it on a promoter is well characterized,
but how TFIIA communicates with TBP in the context of TFIID remains
unclear. TFIIA is generally required for transcription in the presence
of TAFs, yet it is uncertain how the functions of TFIIA and TAFs are
connected. We report here that an important link between TFIIA and
TFIID resides in TAF40. The interaction between TFIIA and TAF40 was
observed both in vivo and in vitro. Thus, the TFIIA-TAF40 interaction
may be directly involved in processes that are TFIIA and TAF dependent.
We also identified a direct interaction between TAF40 and TBP. TAF-TBP
interactions have been identified in other organisms
(
29,
30,
41,
57,
63,
82,
87,
88). In addition,
a 100-amino-acid fragment of
yeast TAF130/145 has been shown to
interact with TBP (
6,
42), and the conserved C-terminal domain
of yeast TAF68
interacts with TBP (
75). However, TAF40 is the
first
full-length TAF that has been shown to interact directly
with yeast TBP
in solution. A TBP-TAF40 interaction is consistent
with studies that
show the human homologue of TAF40, human TAF28,
interacts with human
TBP (
47,
48,
57) and indicates that
this interaction is
conserved from yeast to humans. Human TAF28
has also been shown to
interact with human TAF18, TAF55, TAF100,
TAF135, and the viral
activator Tax (
8,
11,
18,
47,
48,
57). Taken together with
our studies, these results suggest
that TAF40 may have critical
functions in TAF-TAF, TAF-TBP, and
TAF-TFIIA
interactions.
Mutations in TFIIA affect the interaction with TAF40.
The
functional importance for the TFIIA-TAF40 interaction is supported by
mutational studies of the Toa2 subunit of TFIIA in the 4HB domain of
TFIIA. Mutations at the I27 residue of Toa2 (I27A and I27K)
caused a defect in the interaction with TAF40. The loss of interaction
is not due to global changes in the structure of the protein, since
these Toa2 derivatives interact normally with the other subunit of
TFIIA (both in vivo and in vitro), are expressed in cells at levels
similar to that in wild-type Toa2, and are functional for TFIIA-TBP
interactions in vitro. Although the 4HB of TFIIA is not involved in
either TBP or DNA interactions in the crystal structures, a deletion
derivative of human TFIIA lacking two of the helices in this domain is
not responsive to activators in vitro (54). Taken together
with our results, this suggests that the 4HB domain of TFIIA is a
functionally important target that facilitates formation of an active
transcription initiation complex. Since the isoleucine at position 27 of Toa2 is conserved in yeast, Drosophila, and human TFIIA
(23), this surface may play a critical role in the
interaction of the higher eukaryotic homologues of TFIIA and TAF40. It
is interesting to speculate that an interaction between TFIIA and TAF40
would orient the TAF40-TBP interaction on the N-terminal repeat of TBP.
The surface of TBP situated on the same side of the structure as I27 is
the precise location of the altered amino acids in a TBP mutant that is
defective in TFIID formation in vivo (71).
The I27A- and I27K-substituted derivatives displayed mutant growth
phenotypes and defects in transcription in vivo. A complete
cessation
in transcription was not expected, since the mutants
support cell
viability. Transcriptional defects observed in previous
studies in
which TFIIA or TAF40 was inactivated (
35,
44,
53,
61) are
comparable to those reported here. It is clear that
inactivation of
TFIIA does not typically cause dramatic transcriptional
effects. In our
TFIIA mutant strains, it is likely that disruption
of TFIIA-TAF40
interactions could be compensated by redundancy
in other interactions
that contribute to formation of the TBP-TFIIA-TAF40
complex. Important
interactions for complex stability may arise
from TFIIA-TAF40 contacts,
TAF40-TBP contacts, TFIIA-TBP contacts,
and interactions between DNA
and both TFIIA and TBP. This idea
is supported by our in vitro
observations in which the Toa2 mutant
proteins are functional in the
enhancement of the DNA-TBP-TFIIA
complex formation by TAF40. Further
support for redundant interactions
between TFIIA and TFIID is provided
by the observation that TFIIA
mutants defective for both the TAF40 and
TBP interactions are
not viable. In addition, the importance of
compensatory interactions
in the TFIIA-TBP-DNA complex is also
demonstrated by recent reports
showing that mutations in the region of
TFIIA that binds DNA can
suppress mutant phenotypes of TBP
alleles with DNA binding defects
(
53). Moreover, mutations
in Toa2 at glycine 30 (G30) also suppress
the TBP mutant defective for
DNA binding and yet, G30 mutants
have no effect on in vitro
interactions involving TBP, TFIIA,
and DNA (
53). This
result suggests that G30 substitutions can
compensate in vivo by
increasing interactions with another component
of the complex. G30 is
located near the hydrophobic patch of Toa2
and directly abuts I27. It
is interesting to speculate that mutations
at G30 have the potential to
alter the TFIIA-TAF40
interaction.
TAF40 may act as a communicator between TFIIA and TFIID.
The
association between TFIIA and TAF40 is particularly noteworthy because
it supports a growing body of evidence that indicates that
transcriptional activity mediated by TAFs is dependent on TFIIA. TFIIA
is required for in vitro transcription reactions when TAFs are present
(reviewed in reference 27). TFIIA has also been shown to
induce a conformational change in the TFIID complex bound to a
promoter. Specifically, the presence of TFIIA extends the footprint of
TFIID downstream of the transcriptional start site and alters the
cross-linking pattern of several TAFs to promoter sequences (14,
15, 20, 64). It has also been shown that an interaction between
TFIIA and TFIID results in the generation of a productive form of TFIID
that is capable of stably interacting with the promoter
(72). Moreover, under certain conditions, TAFs have been
shown to inhibit the ability of TBP to bind DNA (42, 43, 63,
83). Yet the addition of TFIIA reverses this TAF inhibition
(42, 69).
The identification and characterization of an interaction between TAF40
and TFIIA is a first step in determining the mechanistic
requirement
for TAF40 in transcription. It is clear that TAF40
performs an
essential and nonredundant function in yeast cells,
since it is
required for cell viability (
38) and TAF40 inactivation
appears to affect transcription from RNA Pol II promoters in yeast
(
44,
61). To date, TAF40 appears to be a component
specific
to the TFIID complex (
25,
44,
62,
65), and thus
the TFIIA-TAF40
interaction has the potential to serve as a critical
link between
TFIIA and TFIID functions in
vivo.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant GM56884 to L.A.S.
We are indebted to Kevin Lumb for the Fos AD DNA and for critical
reading of the manuscript. We also thank Zarmik Moqtaderi for various
TAF DNAs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, Colorado State University, Fort
Collins, CO 80523-1870. Phone: (970) 491-5068. Fax: (970) 491-0494. E-mail: lstargel{at}lamar.colostate.edu.
| |
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Molecular and Cellular Biology, March 2001, p. 1737-1746, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1737-1746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
