(Received for publication, August 22, 1996, and in revised form, January 3, 1997)
From the Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021
We previously identified three TATA-binding protein (TBP) point mutations (L114K, L189K, and K211L) that have severe effects on transcriptional activation by acidic activators, but no effect on basal transcription, in a yeast-derived TBP-dependent in vitro transcription system (Kim, T. K., Hashimoto, S., Kelleher, R. J., III, Flanagan, P. M., Kornberg, R. D., Horikoshi, M., and Roeder, R. G. (1994) Nature 369, 252-255). These activation defects were also demonstrated in vivo in yeast cells (Lee, M., and Struhl, K. (1995) Mol. Cell. Biol. 15, 5461-5469). Here, the transcriptional activities of these and other TBP mutations were examined in human by both in vitro and in vivo assays. Mutations L189K and E188K, which lie in the second stirrup region of TBP, show defective activation by acidic activators both in yeast and human. Somewhat surprisingly, mutations L114K and K211L have almost no demonstrable effect on activation by acidic activators in human, in contrast to their severe effects on defective activator responses in yeast. The implications of these results for TBP structure and function are discussed.
TBP1 has been proposed to be the direct or indirect target of several upstream activators in addition to playing a crucial role in basal transcription (reviewed in Refs. 1 and 2). Amino acid sequence comparisons of TBP species from yeast to human have revealed a highly conserved carboxyl-terminal domain of 180 amino acids and a variable amino-terminal domain (reviewed in Ref. 3). The conserved carboxyl-terminal core is known to be sufficient for binding to the TATA box and for basal transcription in vitro (reviewed in Refs. 1-3). Despite striking structural and functional similarities among eukaryotic TBPs, yeast cells are not viable when yeast TBP (yTBP) is replaced with human TBP (hTBP) (4-6). One possibility is that TBP might be involved in species-specific interactions with other transcription factors. However, several other studies showed that yTBP can support both basal and activated transcription by acidic activators in a human in vitro system and that hTBP can mediate responses to acidic activators in yeast cells (7, 8). This interchangeability of yeast and human TBPs argues against species-specific interactions, at least for the response to acidic activators.
Despite this functional interchangeability, hTBP is less active for the response to acidic activators than yTBP in yeast based on levels of reporter gene expression both in vivo and in vitro (7, 8). yTBP and hTBP also differ in another respect. The hTBP is found in a very stable complex (TFIID) containing both TBP and TBP-associated factors (TAFs) with coactivator functions (reviewed in Refs. 1, 2, and 9). In contrast, although yeast contains homologous TAFs that can be isolated in association with yTBP (10, 11), the interactions are apparently much weaker than those reported for the human and Drosophila counterparts. Moreover, although TAFs appear to be obligate coactivators for the in vitro function of activators in human and Drosophila systems (Refs. 12 and 13; reviewed in Refs. 1, 2, and 9), recent studies of yeast TAFs suggest that they are not generally required for the several activators tested (14, 15). In this case activation may be more dependent upon other coactivators, such as components of the holoenzyme mediator complex (reviewed in Ref. 16) or the ADA complex (reviewed in Ref. 17). General coactivators other than TAFs have also been reported in the human system, although they are jointly required with human TAFs for activation (18-20). Given apparent differences in requirements for coactivators (especially TAFs) in yeast and human, one might expect variations in activation mechanisms and in the importance of the well documented interactions of activators with general factors such as TBP (reviewed in Ref. 21) and TFIIB (22).
We previously used a series of point mutants in the conserved carboxyl-terminal core of yeast TBP to define residues important specifically for GAL4-VP16-dependent transcription (23). Mutants L114K, L189K, and K211L were found be defective for the response to GAL4-VP16, but nonetheless showed normal basal transcription by RNA polymerase II. A more detailed analysis revealed that activator-induced TFIIB recruitment to (or stabilization within) the preinitiation complex is disrupted by TBP mutations that impair its interaction with VP16 (L114K), TFIIB (L189K), or the TATA element (L114K and K211L). Focusing on these mutations, the objective of the present study was to identify TBP interactions important for transcriptional activation by acidic activators in human as well as yeast, and possible differences in the two systems. In contrast to the behavior of mutants L114K, L189K, and K211L in yeast, where all are severely defective for activator function, two (L114K and K211L) mediated normal activation responses and another (L189K) showed only a modest defect in human systems.
Transcription factors TFIIA, TFIIE/F/H, and USA were fractionated from HeLa nuclear extracts by phosphocellulose (P-11) chromatography (24) and further purified as described (19). RNA polymerase II was purified from HeLa nuclear pellet extracts by chromatography through heparin-Sepharose, DEAE-cellulose, and Mono Q columns (25). Flag-tagged TFIID was purified by affinity chromatography (18) and was heat-treated for 5-10 min to inactivate endogenous TBP activity at 47 °C as described (26). Recombinant TFIIB and TBPs were expressed as hexahistidine fusion proteins from the T7 polymerase plasmid pET11d in Escherichia coli strain BL21(DE3)pLysS and were purified by nickel affinity chromatography (27, 28). GAL4-VP16 was expressed in E. coli and purified as described before (23). In vitro reconstituted transcription was performed and analyzed as described before (19, 23, 30, 31).
In Vivo Altered-specificity TBP AssayHeLa cells were
transfected with a c-fos reporter plasmid, TBP and GAL4-VP16
expression plasmids, and an -globin internal reference plasmid (32).
After 48 h, RNase protection analysis was performed to quantitate
accurately initiated transcription. Site-directed mutagenesis was used
(33) to introduce the triple amino acid altered-specificity
substitutions (8) into TBP, as well as specific substitutions at the
Leu-114, Glu-188, Leu-189, or Lys-211 residues (23).
The previously analyzed TBP mutations, which selectively abolish
VP16-activated transcription in yeast (23), occur at residues that are
conserved in yTBP and hTBP and located in the conserved carboxyl-terminal domain (Fig. 1A). The
corresponding mutant (L114K, L189K, and K211L) and wild-type yTBPs were
expressed as hexahistidine fusion proteins in E. coli. The
expressed TBPs were purified by nickel affinity chromatography (Fig.
1B) and then employed in various in vitro
transcription assays.
To determine the transcription activities of TBP mutants in human, we
utilized an in vitro transcription system that was
reconstituted with HeLa cell-derived general factors (TFIIA, TFIIB,
TFIIE/F/H, RNA polymerase II, and TBP), the USA cofactor fraction, and
a heat-treated TFIID (Fig. 2). Heat treatment of TFIID
selectively inactivates TBP, leaving a TAF population that, with
ectopic TBP, can mediate activator function (albeit at a level lower
than that observed with intact TFIID)
(26).2 The template used for this analysis
contains five GAL4 binding sites preceding the HIV TATA element fused
to a G-less cassette (Fig. 2A). As shown in Fig.
2B, in the absence of recombinant TBP, no specific
transcription could be detected with general factors and heat-treated
TFIID either alone (lane 1) or in combination with either
the acidic activator GAL4-VP16 or the USA fraction (lanes 2 and 3). A low level of transcription was detected when both
GAL4-VP16 and USA were present (lane 4), probably due to a
trace amount of TBP contamination in the transcription system (19). In
the presence of recombinant wild-type TBP, general factors, and
heat-treated TFIID, a low level of basal transcription was observed
(lane 5). This basal transcription was unaffected by the
sole addition of GAL4-VP16 (lane 6) or USA (lane
7), but both together gave a significant activation (lane
8). This marked activation by GAL4-VP16 was not observed in the
absence of a heat-treated (and near homogeneous) TFIID
preparation,2 which presumably supplies essential TAFs that
can reassociate with TBP. These results indicate that transcription in
this human in vitro reconstituted assay system is dependent
upon exogenous TBP.
Using the TBP-dependent human in vitro system
described above, we analyzed the response of activation-defective TBP
mutants (23) to acidic activators (Fig. 3). Fig.
3A shows an analysis of transcription with different
concentrations of TBP in this reconstituted transcription system.
Additionally, we analyzed the response of TBP mutants (23) to GAL4-VP16
using non-saturating levels of TBPs (Fig. 3B). While
comparable to wild-type TBP with respect to basal activity, the L189K
mutant was defective for activation (lanes 5 and 6 versus lanes 1 and 2). This is consistent with the
effect of the L189K mutation in yeast, although the defect was much
less severe in the human system as compared with the yeast system.
Somewhat surprisingly, mutants L114K and K211L, which were also
severely defective for activated (but not basal) transcription in yeast
(23), supported activated transcription by GAL4-VP16 as efficiently as
did wild-type TBP. These contrasting activities of TBP mutants in the
yeast versus the human system, including the moderately
defective activation response with L189K in the human system, were also
observed with different acidic activators (e.g.
GAL4-AH).2
The contrasting activities of TBP mutants in the yeast
versus the human system might be due to the different
promoters used in the respective assays. The HIV promoter was used in
the human system, whereas CYC1 was used in the previous yeast
transcription assay (23). To rule out this possibility, we determined
the transcription activities of three TBP mutants in the yeast in vitro system with templates containing the HIV human promoter (Fig. 4). In this case, all the mutants (L114K, L189K,
and 211L) were shown to be severely defective for GAL4-VP16 activation. These results indicate that the differences in the ability of mutant
TBPs to mediate activation in yeast versus human systems are
not due to the different promoters used to perform the assays and
suggest, along with the data in Fig. 3, that certain interactions might
be different for the function of acidic activators in yeast and human.
The contrasting responses by TBP mutants appear to be specific for
activated transcription, since all of the 34 original mutant TBPs
tested (see Ref. 23) showed either undetectable activities or basal
activities comparable to those displayed by wild-type TBP in both human
(Table I) and yeast (23) transcription systems.
|
To further examine the contrasting responses with TBP, wild-type yTBP
and hTBP were compared for their ability to support activated
transcription in the TBP-dependent human and yeast in vitro systems. Wild-type yTBP was indistinguishable from wild-type hTBP in support of activation by GAL4-VP16 in the human in
vitro system (Fig. 5B). In contrast,
wild-type hTBP supported the response to acidic activators much less
efficiently than wild-type yTBP in the yeast transcription system (Fig.
5A). This is consistent with the defective response of TBP
mutants only in yeast (see Figs. 3 and 4). Apparently, the human
transcription system is less sensitive to changes in the structure of
TBP than is the yeast transcription system.
Next we assessed the in vivo significance of the in
vitro results described above by using an altered-specificity TBP
in transfection assays (32). Various point mutations (L114K, L189K, and
K211L) were introduced into a TBP derivative that can recognize an
altered TATA box (TTAAA) in a c-fos promoter
containing four GAL4 DNA binding sites (Fig.
6A). Individual TBP and GAL4-VP16 activator proteins were coexpressed by transfection with c-fos
reporter constructs in human (HeLa) cells. Transcription from the
c-fos reporter was measured by RNase protection analysis
with normalization to transcription from an
-globin internal control
plasmid (Fig. 6B). Expression of each TBP mutant was
determined by immunoblot analyses from transfected cells, and the
amounts of the TBP expression plasmids used for transfection were
adjusted to give the same level of expression for each.2
Under these conditions, and consistent with the in vitro
results, mutants L114K and K211L supported transcriptional activation
by GAL4-VP16 as well as did wild-type TBP (Fig. 6B). In
contrast the mutation L189K significantly (over 2-fold) reduced the
ability of TBP to respond to GAL4-VP16 in human cells (lane
2). These results support the conclusion that TBP shows
contrasting activation responses in yeast and human and that the
Leu-189 region plays an important role in transcriptional
activation.
In the three-dimensional TBP structure (34, 35), the L189K mutation
maps within the second stirrup region that connects strands S2 and S3
(Fig. 8). To further assess the important role of this region in
transcriptional activation in the human system, we tested a mutation
(E188K) in an adjacent residue (Glu-188) whose side chain points in the
same general direction as the Leu-189 residue. In both the in
vivo and in vitro assay systems described above, mutant
E188K showed a defective response to GAL4-VP16 that was considerably
greater than that observed for L189K (Fig. 7). In
conjunction with the less dramatic but highly reproducible effect of
the L189K mutant, this compromised activation response by E188K
indicates that the second stirrup region of TBP is critical for
transcriptional activation by acidic activators in the human system.
Here we describe TBP mutants that are defective in transcriptional
activation in both yeast and human systems. These mutations (L189K and
E188K) map within the second stirrup region of TBP connecting strands
S2 and S3
in the three-dimensional TBP structure (34, 35) (see Fig.
8). Importantly, the co-crystal structure of the
TBP·TFIIB·DNA ternary complex shows that these mutations are
located in the TBP·TFIIB interface where the basic surface of TFIIB
interacts with the acidic second stirrup region of TBP and the
negatively charged phosphodiester backbone of DNA (35). Consistent with
this, the L189K mutation was previously demonstrated to be defective
both in TFIIB interactions and in activator-induced TFIIB recruitment
(23, 28, 36). Based on these results, it was proposed that activators
may induce qualitative or quantitative alterations in TFIIB binding to
TBP (or TFIID)·promoter complexes through interactions with either
TFIID or TFIIB, or possibly through simultaneous interactions with both
(22, 23, 28, 36). The activator could either stabilize, or effect the
de novo assembly of, an activation-specific
TBP(TFIID)·TFIIB·promoter complex (e.g. one with a
specific conformation), thereby enhancing the ability of
TBP(TFIID)·TFIIB·promoter complexes to interact with downstream general factors (TFIIE, -F, and -H and RNA polymerase II) in
preinitiation complex assembly (see Refs. 23 and 36 for detailed
discussions). Thus, these activation-defective TBP mutants should be
very helpful in gaining further insights into dynamic structural
changes in promoter complexes during preinitiation complex assembly and
function, and especially the activation-specific
TBP(TFIID)·TFIIB·promoter complex.
While TBP mutants L189K and E188K are defective in activation both in yeast and human, somewhat surprisingly, mutants L114K and K211L, which do not mediate normal activation in yeast (23), support activated transcription by acidic activators as efficiently as wild-type TBP. These contrasting activities of TBP mutants for activator function in the yeast versus the human system are also observed with some other TBP mutants (37, 38), including a K138T/Y139A mutant (29) that cannot interact with TFIIA. These mutants can support transcriptional activation in human but are severely defective for activation responses in yeast.3 Interestingly, all the TBP mutants (23, 37, 38) showing contrasting activation responses have been proposed to affect TBP-TATA element interactions. Since TFIIA can stabilize the interaction of TBP on the TATA element, the K138T/Y139A mutation (29) could also affect the formation of an activation-specific TBP·promoter complex. Thus, one implication from these TBP mutational analyses is that the TBP-TATA element interaction (23, 29, 37, 38) is relatively less important for transcriptional activation in human, compared with the direct interaction of TFIIB with TBP through the second stirrup region.
Based on the contrasting activities of TBP mutants for activation in yeast versus human systems, there may be activation-specific TBP interactions, which are not conserved from yeast to human or which are not essential in the human systems analyzed (e.g. because of redundant or alternative activation mechanisms). It is known that transcriptional stimulation by activators in yeast and human is dependent upon coactivators distinct from the minimal basal factors (reviewed in Refs. 1-3, 16, 17, and 39). In human, both TBP-associated TAFs (reviewed in Refs. 1 and 9) and USA-derived components (18-20) are obligatory for activation in vitro; these components, as well as basal factors that include TFIIB (22), have been identified as direct targets for activators, reflecting increased recruitment of TFIID, TFIIB, and other general initiation factors. Studies in yeast have identified TBP-interacting TAFs, which can facilitate activation function in vitro (10, 11), as well as a complex of other coactivators (SRBs and other genetically defined coactivators) that form part of a holoenzyme complex with RNA polymerase II (reviewed in Refs. 16, 17, and 39). Importantly, both in vitro (40, 41) and in vivo (14, 15) studies have shown that the yeast TAFs may not be generally required for activation in yeast, in sharp contrast to their critical role in human systems. Possibly related, the TBP-TAF interactions in yeast appear much weaker than those reported for human TFIID. Mechanistic studies in yeast have shown that activators can interact directly with coactivators to effect holoenzyme recruitment (42; reviewed in Ref. 16) and that TBP recruitment, potentially by direct activator interactions, can also be limiting for activation in yeast (reviewed in Ref. 39). Given that activation through multiply bound activators most likely involves interactions with several of many potential targets in the general transcription machinery (43, 44), the variable effects of TBP mutants L114K and K211L in yeast versus human may simply reflect differential utilization of various activator-coactivator-basal factor interactions and, especially, variations in requirements for the well documented interactions of activators with TBP in yeast and human (reviewed in Ref. 21).
We thank W. Tansey and W. Herr for the altered-specificity TBP constructs, and S. K. Mahanta and J. Strominger for sharing unpublished data.