(Received for publication, January 19, 1996; and in revised form, February 26, 1996)
From the
The general transcription factor IIA (TFIIA) binds to the TATA
binding protein (TBP) and mediates transcriptional activation by
distinct classes of activators. To elucidate the function of TFIIA in
transcriptional activation, point mutants were created in the human
TFIIA- subunit at positions conserved with the yeast homologue. We
have identified a class of TFIIA mutants that stimulate TBP-DNA binding
(T-A complex) but fail to support transcriptional activation by several
different activators, suggesting that these mutants are defective in
their ability to facilitate an activation step subsequent to TBP
promoter binding. Point mutations of the hydrophobic core of conserved
residues from 65 to 74 resulted in various activation-defective
phenotypes. These residues were found to be important for TFIIA
-
interactions, suggesting that
-
interactions are
critical for TFIIA function as a coactivator. A subset of these
TFIIA-
mutations disrupted transcriptional activation by all
activators tested, except for the Epstein-Barr virus-encoded Zta
protein. The
Y65F,
W72A, and
W72F mutants mediate Zta
activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation.
The
W72A mutant failed to stimulate TFIID-DNA binding (D-A
complex) but was able to form a complex with TFIID and DNA in the
presence of Zta (Z-D-A complex). Thus, the ability of Zta to activate
transcription with
W72A appears to result from a unique ability to
form the stable Z-D-A complex with this mutant. Our results show that
different activators utilize the general factor TFIIA in unique ways
and that TFIIA contributes transcription activation functions in
addition to the facilitation of TBP-DNA binding.
Eukaryotic transcriptional activators stimulate the assembly of
general transcription factors into a stable preinitiation complex at
the start site of class II promoters(1, 2) . The
binding of the general transcription factor IID (TFIID) ()to
the TATA box nucleates the formation of the preinitiation
complex(3, 4) . TFIID consists of TATA binding protein
(TBP) and TBP-associated factors (TAFs), which are essential for the
reconstitution of activated transcription in
vitro(5, 6, 7, 8) . Both TBP
and TAFs have been shown to interact directly with different classes of
transcriptional activation
domains(9, 10, 11, 12, 13, 14, 15, 16) .
Several experimental approaches indicate that binding of TBP to the
promoter is rate-limiting in vivo and that activators that
interact with TBP stimulate this step in preinitiation complex
formation(17, 18, 19, 20, 21, 22) .
TBP binding to the TATA box can be stimulated and stabilized by the
general transcription factor IIA
(TFIIA)(3, 23, 24) . TFIIA has only a modest
effect on basal transcription reconstituted with TBP and a minimal set
of general transcription factors(25) . In contrast,
activator-regulated transcription reconstituted with TFIID (TBP plus
TAFs) is strongly dependent on
TFIIA(23, 26, 27, 28, 29) ,
suggesting that a functional interaction exists between TFIIA,
activators, and the TAFs. The assembly of a TFIIATFIID promoter
complex has been shown to be rate-limiting in in vitro transcription reactions, and the acidic activator GAL4-AH was able
to stimulate this rate-limiting step (30) . The Epstein-Barr
virus-encoded lytic activator, Zta, has also been shown to stimulate
the formation of a TFIIA
TFIID promoter complex that is dependent
on the TAFs and the activation domain of Zta (31, 32) . Thus, it is likely that some activators can
stimulate TBP binding by enhancing an interaction between TFIID and
TFIIA.
How TFIIA mediates activator function is not entirely clear.
TFIIA may function by directly stimulating TBP binding to the TATA
box(3, 4, 24, 33) . In addition,
TFIIA has been shown to compete with repressors for binding to TBP.
Thus, TFIIA has been implicated as a derepressor that prevents
nonproductive preinitiation complex
formation(23, 34, 35, 36, 37, 38, 39) .
TFIIA induces a conformational change in TBP (33) and interacts
with the repeat of lysine residues on helix H2 of yeast
TBP(40) . This same basic region has been implicated in the TBP
interaction with the adenovirus E1A transactivator(10) .
Mutations in this region of TBP were also defective for transcriptional
activation by acidic activators and were reduced for their ability to
bind TAF250(41) . Thus, TFIIA interacts with a
region of TBP critical for multiple activator and TAF functions.
The
cDNAs encoding both TFIIA subunits were initially isolated from yeast,
and each subunit, TOA1 and TOA2, is required for cell viability (42, 43, 44) . Subsequently, human and Drosophila TFIIA were purified as three polypeptides (,
, and
) with molecular weights of 35, 19, and 12 kDa,
respectively(45, 46, 47) . Both the human and Drosophila 35-kDa (
) and 19-kDa (
) TFIIA subunits
share sequence similarity to the amino and carboxyl-terminal ends of
TOA1 (
), respectively, and are encoded by a single gene
(called
)(45, 46, 47) . The
nonconserved spacer domain of TOA1 (
) is dispensable for
yeast cell viability(48) . Yeast depleted of TFIIA have normal
RNA polymerase (pol) I and III activity, yet have reduced pol II
transcription in vivo(48) , indicating that TFIIA is
primarily a pol II transcription factor.
Isolation of the cDNA
encoding the human and Drosophila 12-kDa () TFIIA
subunit, a protein 58% homologous to TOA2 (
), allowed the TFIIA
activities to be reconstituted from recombinant
components(27, 28, 29, 49, 50) .
Using TFIIA-depleted extracts, both basal and activated transcription in vitro required the addition of recombinant TFIIA
(rTFIIA)(27, 50) . Transfection of both human TFIIA
cDNAs in HeLa cells stimulates activator-dependent transcription
4-fold(28) . TFIIA is also required to mediate
activator-dependent transcription in highly purified transcription
systems reconstituted with immunopurified or holo-TFIID
(hIID)(27, 28) . Yeast TFIIA can substitute for human
TFIIA in this system, indicating that TFIIA function is conserved in
eukaryotes(27) . TFIIA-
was selected from a yeast TBP
mutant screen that had a defective acidic activator response, and the
defective phenotype was also rescued by fusion of the mutant TBP to
(51) . These data indicate that the binding of TFIIA-
to TBP may mediate transcriptional stimulation by acidic activators.
Protein interaction assays reveal that TFIIA contacts multiple
components of the preinitiation complex. The Drosophila and
human and
subunits both bind to
TBP(27, 28, 29) , Drosophila
binds to dTAF
110(47) , and human
binds to the VP16 (52) and Zta (27) activators in
an activation domain-dependent manner. Zta was also shown to bind
(52) . The interaction of TFIIA with Zta results in
the enhanced binding of TFIID as a stable promoter-bound complex,
referred to as Z-D-A(32) . We have proposed that TFIIA mediates
an interaction between Zta and TFIID that induces TAFs to contact
promoter sequences downstream of the transcription initiation
site(32) . Most other activators do not stimulate D-A complex
formation as efficiently as does Zta, (
)suggesting that this
type of interaction with TFIIA
TFIID may be limited to a certain
class of activators. While TFIIA appears to be required for all
activators to function, it is not clear whether TFIIA is required in
the same capacity by all activators. Moreover, it is not clear whether
TFIIA has functions in transcriptional activation in addition to the
stabilization of TBP binding. To further examine the role of TFIIA in
the activation process, we engineered point mutants in conserved
TFIIA-
residues and assayed these mutants for TFIIA functions in
transcription activation and stimulation of TBP binding.
Figure 1:
Alanine substitution mutations of
TFIIA-. A, the amino acid composition of human
TFIIA-
is aligned to the yeast
subunit. Absolutely conserved
residues are indicated by a vertical line, while conservative
substitutions are indicated by a double dot. Single human
TFIIA-
residues mutated to an alanine are indicated by a star. Gaps(-) were introduced to maximize the identity
between the two proteins. B, immunoblot of purified and
soluble human TFIIA-
mutant proteins. Equal amounts of purified
human rTFIIA-
mutant proteins were solubilized in D100 buffer and
resolved on 15% SDS-polyacrylamide gel electrophoresis. The immunoblot
was probed with rabbit Abs raised against human rTFIIA-
.
Y3A
was expressed in pQE-9, which has a truncated amino-terminal tag. The
amino-terminal tags did not affect WT TFIIA function in any assays
tested.
Figure 2:
TBP-DNA and protein-protein interactions
of the TFIIA- mutants. A, stimulation of human TBP
binding to the E1B TATA box by the TFIIA-
mutants. Wild type or
mutant TFIIA was used to stimulate human TBP binding to a 29-base pair
oligonucleotide probe containing the adenovirus E1B TATA element. The
complex that contains TBP alone (T) and the one that contains
TBP plus TFIIA (T-A) are indicated by an arrow. B,
interaction of
S-
mutants with GST-
and
GST-
.
S-Labeled input lanes represent approximately
10% of the reaction input (top panel, lane 1).
Binding of WT
to GST-
and GST-
are shown in lanes 3 and 4, respectively. Mutant
S-
bound
proteins were eluted, resolved by 12% SDS-polyacrylamide gel
electrophoresis, and quantitated by PhosphorImager analysis.
mutants that bound at less than 25% of WT (for GST-
) or at less
than 50% of WT (for GST-
) are indicated (-) in a table
format shown in the bottom panel. C, interaction of
S-
mutants with human GST-TBP.
S-labeled
WT
,
D20A,
T64A,
Y65A,
W72A, and
D91A
were used in a GST binding assay with either GST or GST-TBP. Input
represents approximately 10% of the reaction.
S-bound
proteins were eluted and analyzed on 12% SDS-polyacrylamide gel
electrophoresis. Quantitation was performed by PhosphorImager
analysis.
The TFIIA- mutants were assayed for
their ability to stimulate human recombinant TBP binding to a TATA box
containing oligonucleotide in polyacrylamide gel assay EMSA (Fig. 2A). In the absence of TFIIA, TBP binds weakly to
the TATA box probe (T complex; Fig. 2A, lane
1). The addition of WT rTFIIA to TBP resulted in the formation of
a stable TBP
TFIIA DNA complex (T-A complex; Fig. 2A, lane 2). With the E1B TATA
oligonucleotide probe, we found that human T-A complex had a faster
migration than the T complex, suggesting that TFIIA may alter the
conformation of the TBP-DNA complex(33) . The addition of the
various TFIIA-
mutants to TBP produced varying amounts of T-A
complex formation. The TFIIA-
Y65A mutant did not form the T-A
complex at all, while the
Y3A,
D20A,
T64A,
W72A,
and
D91A mutants formed weak T-A complexes (less than 35% of WT) (Fig. 2A). The
W72A mutant was unique in its
ability to form an altered mobility T-A complex in polyacrylamide-EMSA
gels, which was only evident on longer exposures (see below, Fig. 5A).
Figure 5:
Zta overcomes the W72A defect by
stimulating a TFIIA
TFIID promoter complex. A,
W72A
forms an aberrant T-A shift in acrylamide gel EMSA. Longer exposures of
EMSA shown in Fig. 2A revealed that
W72A forms a
unique, slower mobility complex indicated by an asterisk. B,
wild type TFIIA and
W72A were compared under increasingly
favorable conditions for D-A and Z-D-A complex formation. Wild type
TFIIA or
W72A was added at either 1
(50 nM) (lanes 5-8) or 2
(100 nM) (lanes
9-12) concentrations to stimulate hIID (0.2 units) (lanes 3-12) and Zta (16 nM) (lanes 2, 4, 6, 8, 10, and 12)
complex formation. Complexes were formed at either 22 °C for 15 min (top panel) or 30 °C for 60 min (bottom panel).
The shifted D-A or Z-D-A complexes were quantitated by PhosphorImager
analysis and are graphed below each lane as % of the WT
signal.
We have previously demonstrated a direct
protein-protein interaction between S-labeled
and a
GST-TBP fusion protein (27) (Fig. 2C, right
panel). Since the TFIIA-
D20A,
T64A,
Y65A,
W72A, and
D91A mutants all formed weak T-A complexes, these
S-labeled
mutants were tested for their ability to
bind directly to GST-TBP. Both the
D20A and
Y65A mutants were
reduced in GST-TBP binding, 61 and 42% of WT levels, respectively (Fig. 2C, left panel). However, the
T64A,
W72A, and
D91A mutants bound to GST-TBP with a similar
affinity to WT
(Fig. 2C, left panel).
For these activators,
transcription was reduced to less than 5% in extracts depleted of
endogenous TFIIA(27) . Activated transcription could be
restored to undepleted levels by the addition of 0.2 µM WT
rTFIIA(27) . To determine if the TFIIA--containing mutants
(WT
+
mutant) coactivated transcription maximally
at 0.2 µM as did WT rTFIIA in our depleted system,
TFIIA-
mutants were titrated using the Zta and GAL4-AH activators.
We found that, for the panel of TFIIA-
mutants, 0.2 µM also yielded maximal transcriptional activity (data not shown).
For each activator tested in Fig. 3, the addition of 0.2
µM WT rTFIIA to TFIIA-depleted extracts was considered a
100% level of transcription, while TFIIA-depleted extracts were
considered to be a 0% level of transcription. The levels of activated
transcription obtained for a particular TFIIA-
mutant and
activation domain were plotted as percentage of the WT TFIIA signal (% WT) (Fig. 3).
Figure 3:
Mutations of TFIIA- show
activator-specific defects in reconstituted transcription reactions. A, the levels of activated transcription mediated by the
TFIIA-
mutants. Purified activator proteins GAL4-AH, GAL4-CTF, or
Zta were incubated with the indicated TFIIA mutant and TFIIA-depleted
HeLa cell nuclear extracts in in vitro transcription
reactions. The activators used are indicated above each panel. Primer extension reaction products were quantitated by
PhosphorImager analysis and graphed relative to the WT TFIIA signal
(100%) and the TFIIA-depleted extract signal (0%). Wild type or mutant
TFIIA was added to a final concentration of 0.2 µM. The
mean and positive standard deviation are graphed for the TFIIA-
mutants. At least three independent experiments were performed for each
mutant and activator shown. B, T-A complex formation versus the extent of GAL4-AH-activated transcription mediated
by the TFIIA-
mutants. T-A data are the average values from two
independent experiments, and GAL4-AH-activated transcription values are
derived from Fig. 3A; both data are presented side by side. C, the same data as in A but graphed to compare the
transcription levels of a subset of TFIIA-
mutants with different
activators.
Only one point mutation of
TFIIA- was completely defective for all activators tested.
Y65A, which failed to form the T-A complex (Fig. 2A) and had reduced binding to GST-TBP (Fig. 2C), did not mediate transcription for any
activator tested, suggesting that T-A formation is required for
transcription activation (Fig. 3A). However, the
Y6A and
F67A mutants formed T-A complexes at near WT levels
but mediated transcription at just 25% or lower values relative to WT
for all activators (Fig. 3B). This indicates that T-A
formation alone is not sufficient for activator function. Most
significantly, the
W72A mutant supported activation only for Zta,
mediating about 18-fold higher levels of Zta activity compared with
GAL4-AH (Fig. 3C). The
Y3A and
C68A mutants
had similar transcription phenotypes to the
W72A mutant, although
not as dramatic, with at least 2-fold greater activity for Zta compared
with all the other activators tested (Fig. 3C). In
contrast, the
D91A and
E109A mutants revealed an opposite
phenotype, mediating about 2-fold and 3-4-fold, respectively,
higher levels of transcription activation for all the activators
compared with Zta (Fig. 3, A and C). These
results show that mutations in TFIIA-
can affect some activators
more dramatically than others, implying that different activators
utilize TFIIA in distinct activation pathways.
Figure 4:
Conservative substitutions of Y65 and
W72 uncouple T-A formation from transcription activation. A, an immunoblot of equal amounts (200 ng) of TFIIA-WT,
Y65F, and
W72F proteins was probed with rabbit TFIIA-
Abs. B, T-A complex formation of
Y65F and
W72F
proteins. Human TBP was incubated either alone (lane 2) or
with WT TFIIA (lane 3), TFIIA-
Y65A (lane 4),
TFIIA-
Y65F (lane 5), TFIIA-
W72A (lane 6),
or TFIIA-
W72F (lane 7). C, in vitro transcription in TFIIA-depleted nuclear extracts with the
Y65F or
W72F mutants. For the in vitro transcription
reactions, Zta (lanes 1-4), GAL4-AH (lanes
5-8), GAL4-CTF (lanes 9-12), or GAL4-VP16 (lanes 13-18) activator was added to TFIIA-depleted
extracts. 0.2 µM WT TFIIA (lanes 1, 5, 9, and 13), no TFIIA(-) (lanes 2, 6, 10, and 14), TFIIA-
W72F (lanes
3, 7, 11, and 16), TFIIA-
Y65F (lanes 4, 8, 12, and 18),
TFIIA-
W72A (lane 15), or TFIIA-
Y65A (lane
17) mutants were added to TFIIA-depleted reactions. Primer
extension reaction products were quantitated by PhosphorImager analysis
and are shown below each lane as a percentage of the
WT TFIIA signal.
The ability of Zta to uniquely
activate transcription with W72A could be explained if Zta were
able to overcome the defect of
W72A binding to TBP-DNA. Zta has
been shown to stimulate the formation of a stable TFIIA
TFIID
promoter complex (Z-D-A)(32) . We have previously reported that
under limiting conditions TFIID does not form a stable
magnesium-agarose EMSA complex by itself (Fig. 5B, lane 3) or in the presence of either Zta (lane 4) or
TFIIA (lane 5) alone(27, 32) . To examine if
Zta and the TAFs in the TFIID complex are capable of compensating for
the
W72A mutation, we measured D-A and Z-D-A complex formation in
magnesium-agarose EMSA (Fig. 5B). We compared D-A and
Z-D-A formation with the
W72A mutant and WT TFIIA using higher
temperature, longer incubation times, increased amounts of TFIIA
protein, or a combination of the above. Under limiting conditions of 22
°C and 15-min incubations,
W72A forms a much weaker Z-D-A
complex (34%) than does WT TFIIA (Fig. 5B, top
panel, compare lanes 8 and 6 or lanes 12 and 10). Quantitation indicated that TFIIA stimulated
TFIID binding 2-fold (compare lanes 5 and 9 to lane 3), while
W72A did not stimulate TFIID binding (Fig. 5B, top panel; compare lanes 7 and 11 to lane 3). By increasing both the time
and temperature of the incubation reactions, the extent of Z-D-A
complex formed with
W72A increased to levels indistinguishable
from WT TFIIA (Fig. 5B, lower panel, compare lanes 10 and 12). However, these conditions failed to
improve the binding of
W72A to TFIID in the absence of Zta (Fig. 5B, lower panel, compare lanes 11 and 3), while WT TFIIA was improved over 4-fold in D-A
formation (lower panel, compare lanes 9 and 3). These results suggest that
W72A fails to stimulate
TFIID-DNA binding and that Zta can compensate for this defect.
Previous reports identified TFIIA as a critical component in
the rate-limiting steps of activated
transcription(30, 31, 32, 61) . To
better elucidate the function of TFIIA in this process, we sought to
isolate TFIIA mutants that distinguish the transcription function of
several different activation domains. We hypothesized that different
TFIIA surfaces would be required to mediate the activation of different
activators. Since a previous report determined that nearly all in frame
deletions of yeast TFIIA- were nonviable(48) , it appeared
that subtle alterations of TFIIA would be more likely to yield
activator-specific transcriptional defects. To avoid gross alterations
of TFIIA structure, alanines were substituted in single conserved
residues of TFIIA-
in the first panel of mutants. Analysis of
these TFIIA mutant proteins in DNA binding and in vitro transcription assays revealed that TFIIA interacts with different
activators in distinct ways and mediates at least two mechanistically
distinct activator functions.
Transcriptional analysis of the Y6A and
F74A mutants also
suggests that the activation mechanism of GAL4-CTF may be distinct from
the other activators tested. While
Y6A and
F74A were reduced
for most activators relative to WT (the exception being that
F74A
mediates Zta activation), the effects on GAL4-CTF activity were the
most severe (Fig. 3C).
Y6A stimulates T-A
formation at close to WT levels but is substantially reduced for the
formation of Z-D-A complex (data not shown). One likely explanation for
these observations is that
Y6A, and possibly
F74A, fail to
interact with a subset of TAFs important for D-A formation and that
these TAFs are specifically important for GAL4-CTF-mediated
transcription.
Other reports have found mutations of yeast TBP that
specifically disrupt activated but not basal transcription in
vitro(62) . At least one of these mutations was likely to
interfere with TFIIA binding. A more recent transfection analysis of
human TBP mutations revealed that different activators had different
sensitivity to TBP mutations, suggesting they interact with TBP in
distinct ways(41) . While several activation domains responded
differently to various TBP mutants, most of the transcriptional defects
could be correlated with the loss of binding to
TAF250(41) . Similarly, mutagenesis of TFIIB
identified a region of TFIIB that disrupted activated transcription for
two activators but not basal transcription (63) . In contrast,
a mutagenesis analysis of the large subunit of TFIIE did not
distinguish basal from activated transcription nor differences between
two types of activation domains(64) . Thus, the extent to which
mutations of general transcription factors affect different activators
may reflect important differences in activator mechanisms and general
transcription factor functions.
Several
additional activities have been ascribed to TFIIA besides stimulation
of TBP-DNA binding. TFIIA induces a conformational change in
TBP(33) , and it is conceivable that Y65F and
W72F
fail to induce a TBP conformational change necessary for
transcriptional activation by GAL4-AH, CTF, and VP16. The Drosophila
subunit has been shown to bind to
dTAF
110(47) , and mutations in TFIIA-
may
disrupt these interactions, which are critical for transcriptional
activation. The requirement of TFIIA and TAFs in promoter selectivity
also supports the model that TFIIA functionally interacts with
TAFs(65) . Alternatively, coactivators, like PC4 and HMG2, have
been shown to interact with D-A complex
formation(61, 66) , and TFIIA-
W72F and
Y65F
may fail to interact with these coactivators in the preinitiation
complex. TFIIA can also disrupt TBP-specific repressors, like
DR1(36) , and although
W72F and
Y65F form the T-A
complex, they may fail to disrupt specific repressor-TBP interactions.
TFIIA copurifies with a repressor activity specific for TBP and
consensus TATA elements, and our mutations may affect the specificity
of this repressor activity(67) . Additionally, TFIIA makes
direct contact with at least two transcriptional activators (27, 52) , and it is possible that some of these TFIIA
mutations have lost the ability to directly contact specific activators
or coactivators necessary for transcription function. While we have not
determined which of these possible TFIIA interactions have been
disrupted by these mutations, our data strongly suggest that TFIIA
interactions subsequent to T-A formation are essential for
transcription activation.
A general model has emerged
that suggests that TBP binding to DNA is a rate-limiting step affected
by several classes of transcriptional
activators(19, 20, 21, 22) . TFIIA
can stimulate the binding of TBP to DNA, and activators that stimulate
TFIIA binding are predicted to enhance
transcription(24, 32, 51, 60) . The
analysis of TFIIA- mutations presented in this study suggests that
TFIIA not only enhances TBP-DNA binding but qualitatively changes the
preinitiation complex. Our data suggest that TFIIA affects the
recruitment of TAFs and/or coactivators into a transcriptionally active
conformation. Our analysis also indicates that activators function by
distinct mechanisms and that TFIIA plays a central role in
distinguishing the mechanism of different activators.