(Received for publication, May 31, 1995)
From the
RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J.(1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R.(1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP.
Transcriptional activators are sequence-specific DNA-binding proteins, or protein complexes, which contain specific domains dedicated to mediating transcriptional activation. Activation domains are classified according to amino acid composition. Among them, acidic activation domains are highly conserved in function, since they activate transcription from yeast to mammals (Ptashne, 1988). A well-studied example is the herpes simplex virus VP16 activation domain (Gerster and Roeder, 1988; O'Hare et al., 1988; Preston et al., 1988; Triezenberg et al., 1988) whose function requires certain hydrophobic residues, in addition to overall negative charge (Cress and Triezenberg, 1991; Regier et al., 1993).
The mechanism by which the basal transcriptional machinery
increases the rate of initiation upon activation involves
protein-protein contacts between activation domains and specific basal
factors (Ptashne, 1988; Ptashne and Gann, 1990; Roeder, 1991; Gill and
Tjian, 1992). An important yet unresolved question is whether there is
direct interaction between the activation domain and components of the
basal transcriptional machinery (for review of basal factors see (Zawel
and Reinberg, 1993)), or whether additional factors are essential for
this signaling pathway. Biochemical approaches have shown that
activation domains indeed are capable of direct contact with general
factors. For example, the activation domain of VP16 has been shown to
contact directly three general transcription factors, TBP ()(TATA-binding protein (Stringer et al., 1990)),
TFIIB (Roberts et al., 1993) and TFIIH (Xiao et al.,
1994). However, these interactions may not be sufficient for
transcriptional activation, and several lines of evidence suggest that
additional factors are required. For example, TBP is associated with
factors (TAFs) in a complex required for transcriptional stimulation in vitro by several classes of activation domains (Rose and
Botstein, 1983; Pugh and Tjian, 1990; Dynlacht et al., 1991;
Reese et al., 1994). TAFs include several coactivators which
make specific contacts with activation domains to assemble active
transcription machinery (Chen et al., 1994).
Certain other proteins and multiprotein complexes distinct from general factors or TAFs have been shown to be involved in transcriptional activation, such as the PC4 (Ge and Roeder, 1994)/P11 (Kretzschmar et al., 1994) cofactor derived from the earlier described USA (Meisterernst et al., 1991). In yeast, products of the SWI/SNF genes are required for the enhancement of transcription by many transcriptional activators (for review see Winston and Carlson, 1992; Carlson and Laurent, 1994)), perhaps through functional interactions with chromatin (Cote et al., 1994; Imbalzano et al., 1994). Also in yeast are SRB proteins, which interact with the carboxyl-terminal tail of the largest subunit of RNA polymerase II and are required for activated transcription from many promoters (Thompson et al., 1993). These factors, along with several general factors, may constitute a holoenzyme RNA polymerase complex (Kim et al., 1994; Koleske and Young, 1994) which includes a transcriptional stimulatory activity (Kim et al., 1994).
Yeast cofactors required for activation in vitro appeared to be distinct from general transcription factors, and thus were termed mediators (Kelleher et al., 1990) or adaptors (Berger et al., 1990). A genetic selection in yeast (Berger et al., 1992) led to the identification of the putative adaptors ADA2 (Berger et al., 1992) and ADA3 (Piña et al., 1993) which had properties consistent with an adaptor role in transcriptional activation. Recently, employing the same genetic selection, two new genes were identified (Marcus et al., 1994), and one was identical to GCN5, which previously had been found to be required for activated transcription by the yeast activator GCN4 (Georgakopoulos and Thireos, 1992).
Several lines of evidence suggest that ADA2 comprises part of a complex containing additional components. First, mutant strains in either ada3 (Piña et al., 1993) or gcn5 (Marcus et al., 1994) have properties similar to ada2 mutants. Also, yeast two-hybrid analysis and coimmunoprecipitation assays showed interactions between ADA2 and GCN5 (Marcus et al., 1994), ADA3 (Horiuchi et al., 1995), and between ADA2 and the VP16 activation domain (Silverman et al., 1994). One model for the role of such a complex is that it constitutes a physical link to allow productive interaction between the activation domain and the basal transcription apparatus. ADA2 may play a central role in this complex, since it selectively interacts in functional assays with certain acidic activation domains, such as those of VP16 and yeast GCN4, but not with others, such as yeast HAP4 (Berger et al., 1992; Piña et al., 1993). In this report we show specificity in physical associations of ADA2 with activation domains and with TBP. Furthermore, we detect direct interaction between ADA2 and the VP16 activation domain, suggesting that ADA2 may provide determinants within the adaptor complex for interaction with activation domains. These data support a model that the adaptor complex is required for physical interaction of activator proteins with the basal transcriptional machinery.
In the case of purified GAL4-VP16 fusion proteins, binding was performed in a binding buffer with 100 mM NaCl supplemented with purified bovine serum albumin (50 ng/µl) as a competitor. Typically, 600 ng of purified protein was used in each binding reaction. The purification of GAL4-VP16 fusion proteins was described (Berger et al., 1990).
Protein blots were blocked in 5% skim milk, 1
phosphate-buffered saline, and 0.1% Tween20, for 90 min and were then
washed in 1
phosphate-buffered saline, 0.1% Tween20 for 30 min,
with several changes of buffer. The following antibodies were used in
immunodetection:
-ADA2 rabbit polyclonal antisera (Silverman et al., 1994),
-TBP rabbit polyclonal antisera (Reddy and
Hahn, 1991),
-TFIIB rabbit polyclonal anitsera (Pinto et
al., 1994), and
-GAL4 monoclonal antisera (Santa Cruz) was
used to detect GAL4-VP16 fusion proteins. All secondary antibodies used
were conjugated with horseradish peroxidase (Sigma). Immunodetection
was performed using enhanced chemiluminescence (ECL kit; Amersham
Corp.).
Yeast nuclear extract (Lue and Kornberg, 1987) was incubated in separate reactions with equal amounts of beads bearing one of the three activation domains fused to GST, or with beads containing GST alone (Fig. 1A). Proteins remaining bound to beads after extensive washes were eluted with 0.5 M salt buffer, and then both the eluted material and material remaining on beads were analyzed by immunoblotting for the presence of ADA2 (Fig. 1B). The profiles of ADA2 interaction were similar for both 0.5 M-eluted material (Fig. 1B, left panel) and material remaining bound to beads after the salt wash (Fig. 1B, right panel). ADA2 did not bind to the control beads containing GST alone but bound to and eluted from beads containing either GST-VP16 or GST-GCN4. However, ADA2 was not detected in eluate from beads bearing GST-HAP4. Thus, ADA2 physically interacted with acidic activation domains previously found to be functionally dependent on ADA2 activity, i.e. VP16 and GCN4, and did not bind to an acidic activation domain found to be independent of ADA2 activity, i.e. HAP4.
Figure 1:
Binding
of ADA2 to acidic activation domains from herpes virus VP16, yeast
GCN4, and yeast HAP4. A, Coomassie Blue staining of
GST-activation domain fusion proteins. A sample of each bead
preparation is shown that was equal to the amount used for the binding
assay described in panel B. Lane 1, GST alone; lane 2, GST-VP16, VP16 amino acid residues 413-490
(Triezenberg et al., 1988); lane 3, GST-GCN4, GCN4
amino acid residues 9-172 (Hope and Struhl, 1986); lane
4, GST-HAP4, HAP4 amino acid residues 330-554 (Forsburg and
Guarente, 1989). Protein size standards are shown on the left. B, immunoblot analysis of yeast protein associating
with GST activation domain fusions using -ADA2 antisera. Yeast
extract was incubated with GST fusions to activation domains (panel
A). Proteins that bound to, and eluted from, each column were
immunoprobed with
-ADA2 antisera. The left panel shows
protein that eluted from the column in wash buffer containing 0.5 M NaCl. The right panel shows protein remaining on the
column after the high salt wash. Lane 1, GST alone; lane
2, GST-VP16; lane 3, GST-GCN4; lane 4, GST-HAP4; lane 5, (left panel) bacterially expressed ADA2.
Protein size standards are depicted on the
left.
Figure 2:
Binding of yeast ADA2 to GST-VP16 and to
deletion derivatives of VP16. A, schematic of GST
fusions to the VP16 activation domain and its deletion derivatives. The
full-length activation domain of VP16 comprises amino acids
413-490 (Triezenberg et al., 1988), and deleted versions
are shown relative to the full-length activation domain. B, Coomassie Blue staining of GST-VP16 fusion proteins. A sample
from each bead preparation is shown that was equal to the amount used
for the binding assay described in panel C. Lane 1,
GST alone; lane 2, GST-VP16, amino acid residues
413-490; lane 3, GST-VP16, amino acid residues
470-490; lane 4, GST-VP16, amino acid residues
413-470; lane 5, GST-VP16, amino acid residues
413-450. An arrow indicates the expected size of the
413-450 deletion derivative. Protein size standards are shown on
the left. C, immunoblot analysis of yeast proteins
associating with GST-VP16 fusions using -ADA2 antisera. Yeast
nuclear extract was incubated with the GST-VP16 fusions shown in panel B. Proteins which bound to, and eluted from, each column
in wash buffer containing 1 M NaCl were immunoprobed with
-ADA2 antisera. A similar profile of interaction between ADA2 and
subregions of VP16 was seen when the remaining protein bound to beads
was boiled and immunoprobed for ADA2 (data not shown). Lane 1,
GST alone; lane 2, GST-VP16
; lane
3, GST-VP16
; lane 4,
GST-VP16
; lane 5,
GST-VP16
. Protein size standards are depicted
on the left.
Consistent with the previous result (Fig. 1B), ADA2 was detected in eluate from beads containing the full-length VP16 activation domain (Fig. 2C). ADA2 was also found in eluate from beads bearing the VP16 distal region, between residues 470 and 490. The region of VP16 between 413 and 470 also bound ADA2, albeit more weakly than the 470-490 distal region. ADA2 was nearly undetectable in the eluate from beads bearing the proximal VP16 region, between 413 and 450. Thus two regions of VP16 may be important for interaction with ADA2, the region between 470 and 490 and the region between 450 and 470.
Since two distinct regions of the VP16 activation domain
associate with ADA2 in the binding assay, we determined the level of
transcriptional activity of these identical regions of VP16 in yeast.
Gene fusions were generated between subregions of VP16 and the GAL4 DNA-binding domain (amino acids 1-147). The transcriptional
activity of the GAL4-VP16 fusions was tested using a bacterial lacZ reporter gene, whose expression was driven by a GAL4-inducible
promoter (Guarente et al., 1982). As expected,
GAL4-VP16 containing the full-length activation
domain was a potent activator (Table 1). The
GAL4-VP16
fusion was a weak activator.
GAL4-VP16
was intermediate, and the
GAL4-VP16
fusion constituted a strong
activator. Thus the strength of each activation region in the in
vivo assay as GAL4 DNA-binding domain fusions paralleled their
ability to bind to ADA2 as GST fusions (Table 1), suggesting that
strength of activation in vivo is related to the ability of
activator to associate with ADA2 in vitro.
Figure 3:
Transcriptional activation and inhibition
by GAL4-VP16 or deletion derivatives. A, DNA
templates used in the in vitro transcription reactions. The
templates are identical except for the activator-binding sites upstream
of the core promoter, as described (Berger et al., 1990). B, primer extension assay of RNA synthesized in
vitro in yeast nuclear extract. The DNA templates used in each
reaction were: lanes 1-4, gal.3; lanes
5-14, dA/dT. The GAL4-VP16 activation domain fusions used
were: lanes 4 and 5, none; lanes 1 and 12-14, C =
GAL4-VP16; lanes 2 and 9-11, N =
GAL4-VP16
; lanes 3 and 6-8, FL =
GAL4-VP16
. An oligonucleotide containing a GAL4
DNA-binding site was included: lanes 7, 10, and 13, 300 ng; lanes 8, 11, and 14,
600 ng. The amount of GAL4-VP16 protein used in the activation control (lanes 1-3) was 5 pmol, and the amount used in the
inhibition experiment (lanes 6-14) was 10 pmol. The
level of transcription observed at the gal.3 template without addition
of exogenous activator (lane 4) was identical to the level of
transcription observed at a basal template, without upstream binding
sites (data not shown), as shown previously (Berger et al.,
1990).
Purified samples of three GAL4 DNA-binding domain fusions to VP16 were compared in their ability to inhibit activated transcription at the test promoter in vitro. The fusions contained either the full-length activation domain (413-490; FL), the amino-terminal region (413-456; N), or the carboxyl-terminal region (452-490; C). As an initial control to show that all three domains were active in vitro, low noninhibiting concentrations of the GAL4-VP16 fusions were compared in their ability to activate transcription at a promoter containing three GAL4-binding sites (template gal.3 in Fig. 3A). Equal amounts of protein activated transcription to a similar extent (Fig. 3B: compare activated transcription in lanes 1-3 to basal transcription in lane 4).
The
second assay tested the ability of each GAL4-VP16 fusion to inhibit
activated transcription at the test promoter (template dA/dT in Fig. 3A). Activation at the dA/dT template (Fig. 3B, lane 5) is caused by an endogenous
activator in the extract (Lue et al., 1989) and results in
approximately a 5-fold increase in transcription over the basal level (lane 4 and see Berger et al.(1990) for details). In
this assay, as was seen previously (Berger et al., 1990),
GAL4-VP16 strongly inhibited activated transcription at
the dA/dT template (compare lane 6 to lane 5). Also
as seen previously, GAL4-VP16
specifically inhibited
activated transcription when the oligomer containing the GAL4
DNA-binding site was included in the reaction (i.e. transcription in lanes 7 and 8 equal basal
transcription in lane 4). Interestingly, the profile of
inhibition by GAL4-VP16
was comparable to that of FL (lanes 12-14 are similar to lanes 6-8),
while GAL4-VP16
was markedly different (lanes
9-11). In fact, GAL4-VP16
was incapable of
inhibiting activated transcription at all (lanes 10 and 11 are the same as lane 5). Thus, in this inhibition assay,
the carboxyl terminus of VP16 sequestered the adaptor activity. This
correlates with its ability to associate with ADA2 in the GST pull-down
assay (Fig. 2). In contrast, the amino terminus of VP16 failed
to sequester the adaptor activity in the inhibition assay and did not
associate with ADA2 in the GST assay. Thus, these data point to the
carboxyl terminus of the VP16 activation domain as crucial for physical
associations with cofactors required for activation in yeast.
Figure 4:
Direct binding between the VP16 activation
domain and ADA2. A, Coomassie Blue staining of GST
fusion proteins. A sample from each bead preparation is shown that was
equal to the amount used for the binding assay described in panel
B. Lane 1, GST alone. Lane 2, GST-TBP (Eisenmann et al., 1989; Hahn et al., 1989). Lane 3,
GST-TFIIB (Pinto et al., 1992). Lane 4, GST-ADA2
(Berger et al., 1992). Protein size standards are shown on the
left. Apparent difference in mobility of GST protein here and in Fig. 2B relative to size standards (See Blue, Pharmacia
Biotech Inc.) was due to the use of different concentrations of
electrophoresis separating gel. B, immunoblot
analysis of purified GAL4-VP16 proteins binding to GST fusions to ADA2,
TBP, and TFIIB. Purified GAL4-VP16 protein was incubated with the GST
fusions shown in panel A. Protein which eluted from each
column was immunoprobed with -GAL4 monoclonal antisera. Lane input
represents 80% of total material used in GST fusion binding assay. GST
fusions were the same as shown in A. GAL4-VP16 fusion proteins
contain the region of the VP16 activation domain stated for each. C, schematic map of GST fusions to ADA2 and its
deletion derivatives. The full-length ADA2 comprises amino acids
1-434. Deleted versions of ADA2, 1-176 (N),
176-333 (M), and 333-434 (C), are shown
relative to full-length. D, binding of purified GAL4-VP16 to
GST-ADA2. Immunoblot analysis of purified GAL4-VP16 binding to GST
fusions to ADA2 (full-length and deletion derivatives as in panel
C). Material remaining on beads was immunoprobed with
-GAL4
antisera. Lane input represents 80% of total material used in the
binding. Protein size standards are depicted on the
left.
None of the GAL4-VP16 fusion
proteins interacted with GST alone (Fig. 4B). All three
of the GAL4-VP16 fusion proteins interacted with both GST-TBP and
GST-TFIIB (Fig. 4B). These data are consistent with
previous observations that TBP (Stringer et al., 1990) ()and TFIIB (Goodrich et al., 1993; Walker et
al., 1993) bind to the VP16 activation domain and to both amino-
and carboxyl-terminal subregions.
GAL4-VP16 bound to the beads bearing GST-ADA2 (Fig. 4B, top panel). GAL4-VP16
also interacted
with GST-ADA2 (Fig. 4B, lower panel). In
contrast, GAL4VP16
interacted poorly with
GST-ADA2 (Fig. 4B, center panel). These data
indicate that the VP16 activation domain can interact directly with
ADA2. The results also show that the carboxyl terminus of VP16
interacted with ADA2, in agreement with the GST-VP16 binding data (Fig. 2C) and GAL4-VP16 inhibition assay (Fig. 3B).
Thus, ADA2 interacts directly with the VP16 activation domain. To determine the region of ADA2 which interacts with VP16, GST fusions were prepared containing subregions of ADA2 (Fig. 3C). Bead preparations were incubated with purified recombinant GAL4-VP16 protein. GAL4-VP16 specifically interacted with the amino terminus of ADA2, but not with either the middle region or the carboxyl terminus of ADA2 (Fig. 3D). This result further demonstrates specificity of the interaction between ADA2 and the activation domain of VP16.
Figure 5:
Binding to GST-VP16 of yeast TBP and TFIIB
derived from wild type and ada2 extract. A, immunoblot analysis of TBP and TFIIB in
wild type and ada2
extract. TBP or TFIIB was
probed in wild type or ada2
yeast nuclear
extract using
-TBP antisera (upper) or
-TFIIB (lower) antisera. B, immunoblot analysis of
TBP or TFIIB associating with the VP16 activation domain in wild type
or ada2
extract. Yeast nuclear extract was
incubated with the GST fusions listed (proteins are shown in Fig. 2B), and material was eluted in binding buffer
containing 1 M NaCl. The GST fusions used were: lanes 1 and 4, GST alone; lanes 2 and 5,
GST-VP16, VP16 residues 413-490; lanes 3 and 6,
GST-VP16, VP16 residues 470-490. The extracts used were: lanes 1-3, extract from wild type cells; lanes
4-6, extract from ada2
cells.
-TBP (upper) or
-TFIIB (lower) polyclonal
antisera were used for detection.
This dependence of VP16/TBP interaction on ADA2 could indicate that ADA2 itself is required for activation domain/basal factor association, or the dependence could be an indirect consequence of the absence of ADA2 in the mutant extract. In order to distinguish between these alternatives, we tested the ability of TBP or TFIIB to interact with ADA2 in yeast extract. Beads containing comparable amounts of GST alone, GST-TBP, or GST-TFIIB (Fig. 4A) were incubated with yeast extract, and eluates were immunoblotted to detect ADA2. ADA2 did not bind to GST alone (Fig. 6A, right panel). ADA2 interacted with GST-TBP, but not GST-TFIIB (Fig. 6A, left panel). Another experiment, which included GST-VP16 and GST-HAP4 activation domain fusions as positive and negative controls, respectively, also showed association of ADA2 with GST-TBP, but not with GST-TFIIB (Fig. 6A, right panel). Thus, since ADA2 associated with TBP (Fig. 6A), and since ADA2 was required for VP16 interaction with TBP (Fig. 5B), we conclude that ADA2 itself may be physically required for VP16 interaction with TBP in a yeast nuclear extract.
Figure 6:
Interaction between ADA2 and TBP or TFIIB. A, immunoblot analysis of ADA2 binding to GST-TBP or
GST-TFIIB. Yeast nuclear extract was incubated with the GST fusion
proteins listed (proteins are shown in Figs. 1A and
4A), and protein was eluted in binding buffer containing 1 M NaCl. Either GST alone, GST-VP16 = VP16
residues 413-490, GST-HAP4
= HAP4 residues
330-554, GST-TBP, or GST-TFIIB was used in each incubation. The
GST fusion proteins were normalized relative to one another before use.
The Western blot was immunoprobed with
-ADA2 antisera. B, immunoblot analysis of TBP associating with GST-ADA2.
Bacterial extract containing TBP was incubated with the GST fusions
listed (proteins are shown in Figs. 1A and 4A), and
protein eluting in binding buffer containing 1 M NaCl, or
material remaining on beads, was immunoprobed with
-TBP
antisera.
Since endogenous ADA2 in yeast extract was capable of
interacting with GST-TBP we wished to determine whether ADA2 and TBP
could interact directly in the absence of other yeast proteins.
Bacterially expressed TBP was incubated with GST alone, GST-VP16 as a
positive control, or GST-ADA2, and eluates were immunoprobed for TBP.
The results show that TBP was retained by GST-VP16 affinity matrix, but
not by GST or GST-ADA2 (Fig. 6B). Thus, there was no
interaction between TBP and ADA2 in these conditions, suggesting a
requirement for additional yeast factors. Consistent with these data,
reticulocyte-translated ADA2 and TBP did not coimmunoprecipitate using
either -ADA2 or
-TBP antisera (data not shown).
Previous work demonstrated that the putative adaptors ADA2, ADA3, and GCN5 are required for transcriptional activation. First, mutations in these genes lowered the activity of certain acidic activation domains, both in vivo and in vitro (Berger et al., 1992; Piña et al., 1993; Marcus et al., 1994), and, second, mutations in ADA2 reduced activated transcription without altering basal transcription in vitro (Berger et al., 1992). These functional effects could be a consequence of activation domain interactions with adaptor proteins, or adaptors could have an indirect effect on transcription. To distinguish between a direct or indirect role for ADA2, we have examined interactions between ADA2, activation domains, and basal transcription factors.
The data presented here support the model that ADA2 provides a physical function in transcriptional activation and may provide a physical link between certain activation domains and basal transcription factors. ADA2 interacted with activation domains derived from the transcriptional activators VP16 and yeast GCN4. These activators have been shown to require ADA2 for their function both in vivo and in vitro. A different approach previously showed that the VP16 activation domain associated with endogenous ADA2 in yeast extract (Silverman et al., 1994). In the present study we extended these observations with the finding that ADA2 binds poorly to yeast HAP4, an activation domain that previously was shown to be functionally independent of ADA2 (Berger et al., 1992; Piña et al., 1993). Thus, the specificity shown in this physical interaction assay supports the specificity previously observed in the functional assays. The consistency between these assays lends support to the model that ADA2 has a physical role in the transcription process. Furthermore, the lack of physical interaction between ADA2 and the HAP4 activation domain result raises the possibility that different cofactors or adaptors may be involved in HAP4 interactions with the basal machinery. Additional biochemical and genetic data support the notion that HAP4 uses different cofactors than VP16 or GCN4 (Wang et al., 1995).
The GST pull-down assay indicated a direct interaction between ADA2 and the VP16 activation domain. Interaction between the carboxyl-terminal region of VP16 and ADA2 was shown in two ways: the 470-490 region of VP16 (as a GST fusion) interacted with endogenous ADA2 in yeast extract and, second, ADA2 (as a GST fusion) interacted with the same region of VP16 purified as a GAL4-VP16 fusion. These results indicated that ADA2 provides determinants for direct interaction with VP16. Consistent with these results, we show that the carboxyl region of VP16, but not the amino region, can sequester factors required for activation in a transcriptional inhibition assay in vitro.
Further evidence that ADA2 provides specific
determinants for direct interaction with VP16 was the demonstration
that the amino terminus of ADA2, but not the remainder of the protein,
bound VP16. This observation is particularly important, since a
putative human homologue of ADA2 ()exhibits strong
conservation in the amino terminus. Since VP16 is a higher eukaryotic
protein, it is reasonable that the region of yeast ADA2 that interacts
with VP16 would be conserved in the ADA2 human homologue.
Our data suggest that the role of the adaptor complex may be to promote association of VP16 with TBP. This conclusion rests on two observations. First, VP16 associated with both TBP and TFIIB derived from nuclear extracts. However, binding of TBP to VP16 was not detected in nuclear extracts lacking ADA2. Thus, VP16 association with TBP in this assay was strictly ADA2-dependent. In contrast, TFIIB association with VP16 was only moderately affected in extracts lacking ADA2, which may support the proposal that VP16/TFIIB interaction is direct (Lin et al., 1991; Roberts et al., 1993), although we cannot rule out the possibility that accessory factors are required for VP16/TFIIB interaction in vivo. Second, we detected physical association between GST-TBP and endogenous ADA2 in nuclear extract but not between GST-TFIIB and ADA2. Thus, since ADA2 in nuclear extract bound to the VP16 activation domain, and since ADA2 and VP16 interacted in a binding assay using purified proteins, we conclude that ADA2 may be required for VP16 interaction with TBP. We did not detect interaction between ADA2 and recombinant TBP under conditions where VP16 and TBP interact, leading to the conclusion that ADA2 may not interact directly with TBP. Since both ADA2 and TBP are part of multisubunit complexes, it is possible that one or many additional proteins are required for completion of the interaction pathway between VP16/ADA2 and TBP. These proteins could include ADA3 and GCN5, which form a complex with ADA2 (Marcus et al., 1994; Horiuchi et al., 1995), or TAFs, which occur in a complex with TBP (Reese et al., 1994), or other general factors that associate with TBP, such as TFIIA, which has been shown to be important for activation in the mammalian system (Ozer et al., 1994).
TBP interacted directly with VP16 (Stringer et al., 1990) and other activation domains in vitro, including that of the tumor suppressor p53 (Seto et al., 1992; Liu et al., 1993) and adenovirus E1A (Lee et al., 1991). One explanation for the requirement for ADA2 in yeast may be that adaptor proteins are required for efficient interaction of activation domains and general factors in vivo. Certain other factors have been identified which have important functions in transcriptional activation and, like ADA2, are not basal factors. TAFs, or coactivators, were recognized as essential components of the activation process in vitro but were dispensable for basal transcription. Many of the human and Drosophila melanogaster TAFs have been cloned, sequenced, and shown to interact with activators and basal factors (Dynlacht et al., 1993; Goodrich et al., 1993; Hoey et al., 1993; Ruppert et al., 1993; Weinzierl et al., 1993). TAF40 physically interacted with the carboxyl-terminal half of VP16, between residues 452 and 490 (Goodrich et al., 1993). This region includes both domains (450-470 and 470-490) which interacted with ADA2 in the data reported here. However, our experiments show that ADA2 association with VP16 was necessary for TBP, but not TFIIB, binding to VP16, whereas TAF40 appeared to interact with TFIIB. Also, there is no obvious similarity between TAF40 and ADA2. Thus, their pathways of action may be distinct.
Recent results indicate that, as in higher eukaryotes, TAFs are also associated with Saccharomyces cerevisiae TBP (Hisatake et al., 1993; Poon and Weil, 1993). Since ADA2 appears to be essential for VP16 interaction with TBP, it is possible that ADA2 is a TAF. Cloning and sequencing of TAFs have revealed only limited homology to adaptors, such as the bromodomain shared by yeast GCN5 and TAF 250 (Ruppert et al., 1993).
Recently a family of adaptor proteins has been identified in higher eukaryotes (Arany et al., 1995; Lundblad et al., 1995). These adaptors, CBP and p300, contain striking similarity to a putative zinc-binding motif in the amino terminus of ADA2 (Arany et al., 1994). A 133 amino acid region of CBP containing the ADA2-homologous zinc-binding domain has been shown to be sufficient for CBP interaction with TFIIB (Kwok et al., 1994). ADA2 did not interact with TFIIB in our assays, suggesting that the similar zinc fingers of CBP and ADA2 may have different roles in transcription.
Our data demonstrate that ADA2 can physically interact with activation domains and TBP. In these binding assays ADA2 has an essential role in stabilizing or recruiting TBP interaction with the activation domain of VP16. Further experiments should elucidate the nature of the interaction of ADA2 with VP16 and TBP and how this interaction contributes to transcriptional activation.