From the The Wistar Institute, Philadelphia,
Pennsylvania 19104 and ¶ Department of Biological Chemistry,
UCLA School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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The interaction of the general transcription factor (TF) IIA with TFIID is required for transcription activation in vitro. TFIID consists of the TATA-binding protein (TBP) and TBP associated factors (TAFIIs). TFIIA binds directly to TBP and stabilizes its interaction with TATA-containing DNA. In this work, we present evidence that TAFIIs inhibit TBP-DNA and TBP-TFIIA binding, and that TFIIA stimulates transcription, in part, by overcoming this TAFII-mediated inhibition of TBP-DNA binding. TFIIA mutants modestly compromised for interaction with TBP were found to be significantly more defective in forming complexes with TFIID. Subtle changes in the stability or conformation of the TFIIA-TBP complex resulted in a failure of TFIIA to overcome TAFII-mediated inhibition of TBP-DNA binding and transcription function. Inhibition of TBP-DNA binding by TAFIIs could be partially relieved by limited proteolysis of TFIID. Proteolysis significantly stimulated TFIIA-TFIID-TATA binding in both electrophoresis mobility shift assay and DNase I footprinting but had little effect on complexes formed with TBP. Recombinant TAFII250 inhibits TBP-DNA binding, whereas preincubation of TFIIA with TBP prevents this inhibition. Thus, TFIIA competes with TAFII250 for access to TBP and alters the TATA binding properties of the resulting complex. Transcriptional activation by Zta was enhanced by temperature shift inactivation of TAFII250 in the ts13 cell line, suggesting that TAFII250 has transcriptional inhibitory activity in vivo. Together, these results suggest that TAFIIs may regulate transcription initiation by inhibiting TBP-TFIIA and TBP-DNA complex formation.
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INTRODUCTION |
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Transcription initiation by RNA polymerase II is regulated by the formation of a preinitiation complex consisting of general transcription factors (reviewed in Refs. 1 and 2). The interaction of the TATA-binding protein (TBP)1 with promoter DNA is an early and highly regulated step in preinitiation complex assembly at TATA containing promoters (Refs. 3-8 and reviewed in Refs. 9 and 10). Several factors bind directly to TBP and modulate its ability to form an active preinitiation complex. TAFIIs and TFIIA bind directly to TBP and are required for transcriptional activation from most promoters in vitro (reviewed in Refs. 11-14). Both TFIIA and TAFIIs can interact directly with promoter-specific activators and mediate their stable interaction with TBP and the preinitiation complex (15-20). TAFIIs can bind directly to core promoter sequences and recruit TBP to promoters lacking consensus TATA elements (21-23). TFIIA makes direct contact with DNA sequences upstream of the TATA box of some promoters and stabilizes TBP binding to TATA-containing DNA (24-27). Thus, both TFIIA and TAFIIs can modulate the interactions of TBP with activators, DNA, and other general transcription factors. However, the potential interactions between TFIIA and TAFIIs have been less extensively examined and are likely to be an important component of eukaryotic transcriptional regulation.
TFIID consists of TBP and at least eight TAFIIs (reviewed in Refs. 11 and 14). TAFII250 binds directly to TBP and multiple other TAFIIs, thus serving as a potential scaffold for the multiprotein complex (28, 29). Other TAFIIs can bind TBP directly, including a subset of TAFIIs with homology to histone proteins, which form a histone fold motif (30). Histones have generally been associated with transcriptional repression by limiting activator and general factor access to promoter sequences, and it is possible that some TAFIIs share histone-like features (31). Purified TAFII250 has been shown to inhibit the DNA binding activity of TBP (21, 28, 32). The functional significance of this inhibition has not been well characterized. TAFII250 is identical to CCG1, a gene identified by a mutation that causes arrest in the G1 phase of the cell cycle. TAFII250 has been shown to have both protein kinase and histone acetylase activities, but the functional significance of these activities is not yet clear (33, 34). Mutations in TBP, which abrogate transcription activation function in vivo, were found to disrupt the ability of TBP to bind to TAFII250 in vitro (35). More recent studies in yeast and hamster cell lines indicate that TAFII250 is required for coordinating the interaction between promoter-specific activators and core promoter elements (36-39).
Several lines of evidence indicate that a dynamic interplay between TFIIA and TAFIIs regulates the binding of TFIID to promoter DNA. Cross-linking studies indicate that TFIIA significantly alters the interaction of TAFIIs with promoter DNA (40). TFIIA mediates an activator-dependent conformational change in TFIID that results in the interaction of TAFIIs with sequences near and downstream of the transcriptional initiation site (20, 41-43). The recruitment of TFIIA by an activator significantly enhanced the ability of TFIID to interact with TFIIB, further indicating that TFIIA can alter the properties of TFIID (43). TFIIA can derepress the inhibitory effects of several factors that bind directly to TBP (5, 44-47). The direct binding of TFIIA to TBP is likely to preclude interaction of these inhibitory factors with TBP. The derepression function of TFIIA is mediated by the subunits of TFIIA that contact TBP directly, but these subunits are not sufficient for TFIIA-mediated transcriptional activation function in vitro (48).
The crystal structure of the yeast TFIIA-TBP-DNA ternary complex
reveals that TFIIA makes direct contact with TBP through several
hydrophobic residues in the small subunit of TFIIA (Toa2) (26, 27).
Mutagenesis of the homologous residues in the human TFIIA small subunit
( ) interrupt TFIIA stimulation of TBP-DNA binding and
TFIIA-mediated transcription stimulation in vitro (49).
Interestingly, conservative phenylalanine substitution mutation of
these residues (TFIIA
Y65F and W72F) had little detectable effect
on the formation of a TFIIA-TBP DNA complex but were found to be
defective for transcription activation in vitro (49). The
biochemical basis for these defects was not clear but suggest that
subtle changes in the TFIIA-TBP interface have dramatic effects on
transcription function. In this work, we further investigated the
mechanism underlying the transcription defect caused by these TFIIA
mutations. We showed that these mutants are substantially more
defective in the presence of TAFIIs and that
TAFIIs are generally inhibitory for TBP-TFIIA complex
formation. We also show that the ability of TFIIA to overcome
TAFII-mediated inhibition correlates with transcription
activation function in vitro.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs--
The Escherichia coli
expression constructs for human wild type and mutant TFIIA- (pRSET
A-IIA-
) and TBP were previously described (49). The E. coli expression constructs for human TFIIA-
(pQE-IIA-
)
was described previously (20). Baculovirus expressed human
TAFII250 was kindly provided by R. Tjian (29). Eukaryotic
expression vector for TAFII250 (pCMV-TAFII250)
was a gift from E. Wang, and Zta was expressed from the SV40 promoter in pBXGo.
Protein Preparations--
The pQE-IIA- and pRSET A-IIA-
wild type or mutant constructs were expressed in M15 and BL21 E. coli strains, respectively. Expressed proteins were purified under
denaturing conditions on nickel nitrilotriacetic acid-agarose columns
(Qiagen) and dialyzed into D100 buffer as described (20, 49).
Recombinant TFIIA polypeptides were used at a final concentration of
~0.2 µM for in vitro transcription
reactions. Human recombinant TBP and recombinant Zta proteins were
prepared as described previously (41). The GAL4-AH fusion protein was
purified as described (50). Baculovirus-expressed human
TAFII250 was purified from baculovirus-infected cell
lysates using the 12CA5 monoclonal antibody for affinity purification and peptide elution essentially as described (51). Affinity-purified holo-TFIID was purified as described (51).
DNA Binding Reactions-- Polyacrylamide EMSA conditions for T-A complex formation were described (52). Conditions for Mg2+-agarose EMSA were described (41, 53). For Mg2+-agarose EMSA Zta-hIID-IIA complex formation, 0.2 unit of human IID, 80 nM TFIIA, and 33 nM Zta were used (41). DNase I footprinting reactions were performed as described previously (54). For protease experiments, protein and DNA were preincubated for 30 min at 30 °C before the addition of 40 ng of protease K for 20 min at 30 °C (unless noted otherwise). The relative mobility of the TBP-DNA (T) and TFIIA-TBP-DNA (T-A) complexes varied dramatically in EMSA depending upon the probe used (E1B or E4T) and whether magnesium was included in gels (46, 49). The positions of T and T-A are indicated in each figure.
In Vitro Transcription Reactions--
In vitro
transcription reactions contained 100 ng of G5E4TCAT
template, ~200 ng of purified recombinant-GAL4-AH activator protein,
and 40 µg of TFIIA-depleted HeLa nuclear extract in a 50 µl final
reaction volume incubated for 1 h at 30 °C. Primer extension
reactions were described previously (51). The TFIIA-depleted nuclear
extracts were described (20). Wild-type or mutant pRSET A-IIA- was
added back to transcription reactions as described (49).
Transfection and CAT Assay-- The hamster ts13 cell line was purchased from ATCC. Approximately 5 × 105 cells were transfected in a 100-mM dish using calcium phosphate precipitation with 250 ng reporter, 5 µg of SV-Zta and 30 µg of CMV-TAFII250 (where indicated). Vector DNA was used to compensate for differences in DNA concentration of effector plasmid. Transfected cells were incubated for 8-12 h at 34 °C, washed with phosphate-buffered saline, and then incubated at 34 or 39.5 °C as indicated. Cells were harvested for CAT assay 36 to 48 h post-transfection. CAT reactions analyzed by thin layer chromatography and quantitated with a PhosphorImager 445SI screen. All transfections were repeated at least three times.
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RESULTS |
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Mutations in TFIIA Disrupt Complex Formation with TFIID--
In a
previous study, we examined a large panel of TFIIA- mutants for the
ability to stimulate TBP binding to the adenovirus E1B core promoter
and to mediate activator stimulated transcription in vitro.
We found that alanine substitution of Tyr-65 completely eliminated the
TBP-TFIIA DNA complex (T-A), whereas W72A caused an aberrant migrating
complex in acrylamide EMSA. Alanine substitution of these residues and
neighboring aromatic residues reduced transcription activation by
several activators in vitro (49). To explore the possible
effect of TFIIA mutations on interactions with TAFIIs in
the TFIID complex, we compared the panel of TFIIA mutants for their
ability to support an interaction between the Zta transcriptional activator and TFIID (Fig. 1). Zta
stimulates a highly stable interaction between TFIIA, TFIID, and
promoter DNA (referred to as the Z-D-A complex) that correlates with
transcriptional activation function for a subset of promoters (55).
TFIIA mutants were first tested for their ability to form a stable
complex with TBP (referred to as T-A) on the E4T promoter. Under
limiting conditions, TBP did not form a stable Mg2+-agarose
EMSA complex by itself but forms a weak smear (Fig. 1A, lane 2). The addition of wt TFIIA results in a stable well
resolved T-A complex (Fig. 1A, lane 3). Similar
stable complexes were resolved for most TFIIA-
substitution mutants,
with the exception of
Y65A, which failed to form T-A (lane
10). Several substitution mutations were reduced for T-A complex
formation, and
W72A produced a T-A complex with a slower mobility in
these Mg2+-agarose EMSA (Fig. 1A, lane
13 and data not shown). We previously reported an altered mobility
complex with the
W72A mutant in polyacrylamide-EMSA (49). These
Mg2+-EMSA results are consistent with previous studies
examining the ability of these TFIIA mutants to form a T-A complex with
the E1B 30-base pair oligonucleotide in polyacrylamide gel EMSA
(49).
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Phenylalanine Substitution of Tyr-65 Decreases Stability of
T-A--
The semiconservative substitutions at residues Tyr-65 and
Trp-72 were particularly interesting because these form T-A but were
defective for D-A formation and transcriptional coactivation (Fig. 2)
(49). Based on crystal structure, we would predict that mutations at
these residues should have a primary defect in the interaction with
TBP. To determine whether these TFIIA mutants alter the stability of
the T-A complex, we compared the dissociation rate of T-A formed with
the wild-type and mutant TFIIAs. Dissociation rates were measured in
polyacrylamide EMSA by the addition of excess unlabeled TATA
oligonucleotide competitor to preformed T-A-DNA complexes (Fig.
3A). T-A complex stability was
assayed at increasing times after the addition of competitor TATA
oligonucleotide. We found that Y65F had a significantly increased
dissociation rate relative to wild-type TFIIA because there was an
~8-fold difference in the reduction of T-A complex after 4 h of
oligonucleotide challenge relative to the reduction of complex formed
with wild-type IIA (Fig. 3A, lanes 18 and
19). In contrast, the
W72A mutant showed no significant
change in the dissociation rate relative to wild-type TFIIA after
4 h of oligonucleotide challenge (Fig. 3A). Thus, the
transcriptional defect of
Y65F may be attributed, in part, to
decreasing the stability of the T-A-DNA ternary complex.
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Substitutions at Trp-72 Increase Protease Sensitivity of
T-A--
Mutations at residue Trp-72 produce a stable T-A complex but
often give rise to a species of slower mobility. To explore the possibility that TFIIA-W72A and
W72F may produce alternative conformations in the ternary complex with TBP and DNA, we used limited
proteolysis as a probe of protein structure and stability. Limited
proteolysis has been used previously to map a core domain of TBP and
TFIIA and for evidence that TFIIA induces a conformational change in
TBP (54, 56, 57). Treatment of TBP with protease K resulted in a slight
stimulation of DNA binding and a faster mobility in acrylamide EMSA
(Fig. 3B, lane 1 to lanes 6,
11, and 16). Treatment of T-A complexes formed
with wt TFIIA resulted in a stable faster migrating complex (Fig.
3B, compare lane 2 to lanes 7,
12, and 17). Interestingly, complexes formed with either
W72A or
W72F resulted in nearly complete digestion of the
T-A complex, with an 8-fold reduction of complex formation compared
with wild-type T-A (Fig. 3B). In contrast, the complex formed with
Y65F was as protease-resistant as wt TFIIA (Fig. 3B). Digestion of TBP before DNA binding resulted in the
complete loss of binding activity, similar to what was seen for the
Trp-72 mutants (Ref. 54 and data not shown). These results show that both TBP and TFIIA-TBP assume a stable and protease-resistant conformation once bound to DNA. The
Trp-72 mutants failed to form
the protease-resistant complex and also failed to stimulate transcriptional activation, suggesting that the protease-resistant conformation is important for transcription activation function. We
suggest that
Trp-72 mutants trap TBP in an inactive conformation and
that this conformation may occur under natural conditions in
vivo. Conformational changes in TBP may be the mechanistic basis
for some transcriptional regulatory pathways.
Proteolysis Stimulates TFIID-DNA and TFIID-IIA Binding-- To further explore the effect of TAFIIs on TFIIA-TBP and TBP-DNA binding, we tested the effects of limited proteolysis on the behavior of TFIID in DNA binding reactions with or without TFIIA (Fig. 4A). Despite the large molecular mass of the TFIID complex, TFIID-DNA complexes could be partially resolved in Mg2+-acrylamide EMSA (Fig. 4A, lane 2). The addition of protease K (40 ng) for 20 min resulted in a striking stimulation of DNA binding activity in this assay (lane 3). Some of this stimulation may result from the degradation of TAFIIs that prevent TFIID-DNA from entering acrylamide gels. In contrast, recombinant TBP bound to similar amounts of DNA (lane 6) but was only modestly stimulated by treatment with protease K (lane 7). The addition of TFIIA to TFIID resulted in a significant stimulation of DNA binding (compare lanes 2 and 4). Interestingly, the addition of protease further stimulated the TFIIA-TFIID complex as well as increased the electrophoretic mobility of the complex (lane 5). The addition of TFIIA to TBP stimulated TBP-DNA binding (compare lanes 6 and 8). Protease K treatment increased the mobility of the TBP-TFIIA complex but had only a minor stimulatory effect on the amount of complex formed (lane 9). One interpretation of this result is that protease degrades TAFIIs, which inhibit TFIIA and DNA interactions with TFIID. Curiously, TFIID- and TBP- containing complexes had identical mobilities both before and after protease treatment. Previous work has also found similar mobilities between TBP and TFIID in EMSA, which may be accounted for by the unusual conformational constraints imposed on DNA bound by TBP (41, 53).
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TFIIA Can Prevent TAFII250 Inhibition of TBP-DNA Binding-- Evidence from proteolysis experiments suggests that degradation of TAFIIs may stimulate TFIIA-TFIID-DNA complex formation. To determine if a single TAFII may confer an inhibitory effect on TFIIA association with TBP, we examined the properties of affinity-purified recombinant human TAFII250 in DNA binding reactions with or without TFIIA (Fig. 6). Others have shown that the addition of recombinant TAFII250 to recombinant TBP results in an inhibition of TATA binding activity (28, 32). TFIIA binds directly to TBP and can preclude the interaction of TBP with several transcriptional inhibitors (44-47). To determine if TFIIA could prevent the TAFII250 inhibition of TBP-TATA binding, we initially determined whether addition of TAFII250 could inhibit DNA binding of preformed T-A complexes (Fig. 6). Incubation of TBP with TFIIA resulted in a strong stimulation of DNA binding (Fig. 6A, lane 3). When TFIIA was preincubated with TBP for 15 min followed by the addition of TAFII250, TFIIA-TBP-DNA complex formation was reduced only slightly, indicating that this complex was largely resistant to TAFII250-mediated repression (lane 4). In contrast, if TAFII250 was preincubated with TBP for 15 min followed by the addition of TFIIA, DNA binding was abolished (Fig. 6A, lane 5). A time course of preincubation with TFIIA-TBP-DNA was performed to determine how much time was required to establish a stable TAFII250-resistant TFIIA-TBP-DNA complex (Fig. 6B). We found that simultaneous addition of TAFII250 and TFIIA to TBP and DNA resulted in a significant inhibition of DNA binding (Fig. 6B, lane 4). However, after 15 min of preincubation with TFIIA, TBP, and DNA, a stable complex formed that was resistant to TAFII250 inhibition (lane 6). Thus, TFIIA competes with TAFII250 for interaction with TBP, and the resulting complexes have opposite effects on the DNA binding properties of TBP.
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Mutant TAFII250 Enhances Transcription By an Activator in Vivo-- To determine what effect TAFII250 inhibition of TBP-DNA or TBP-TFIIA complexes have on transcription activator function, we assayed Zta-mediated transcriptional activation in the ts13 cell line at permissive and nonpermissive temperatures (Fig. 7). The ts13 cell line contains a mutated TAFII250 gene, which results in G1 cell cycle arrest at nonpermissive temperatures (39.5 °C). Transfection of Zta resulted in ~15-fold level of activation in the ts13 cell line at the permissive temperature (34 °C) on the Z5E4TCAT reporter plasmid. In contrast, transfection of Zta resulted in a ~42-fold level of activation in ts13 cells at the nonpermissive temperature (Fig. 7A). Activator levels were not effected by temperature shift in ts13 cells, and a similar temperature shift had no effect on transactivation levels in control baby hamster kidney cell lines (data not shown). Furthermore, a temperature shift resulted in a slight decrease in basal level transcription, indicating that temperature shift by itself did not produce a generalized increase in CAT activity (data not shown). Similar increased transcriptional effects were observed with TAFII250 inactivation in ts13 cells using a different reporter promoter (BHLF1) with Zta or a different activator (GAL4-VP16) with the G5E4TCAT reporter (data not shown). This suggests that the transcriptional effect is not specific for the Zta activator or the template. To determine if this transcriptional enhancement was a result of the mutated TAFII250 gene, wild-type TAFII250 was cotransfected with Zta and reporter plasmids in ts13 cells (Fig. 7B). Cotransfection of wild-type TAFII250 reduced Zta activation levels at the nonpermissive temperature to levels similar to that observed at the permissive temperature. Thus, introduction of wild-type TAFII250 partially reversed the enhancement of activation caused by temperature shift, suggesting that mutant TAFII250 is responsible for the Zta-dependent transcriptional enhancement. Although these transfection experiments do not address the direct role of TAFII250 in transcription function, they do provide indirect evidence to suggest that TAFII250 can inhibit transcription function by an activator in vivo.
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DISCUSSION |
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Although the interaction of TFIIA with TBP has been well characterized, relatively little is understood of how TAFIIs may alter the interaction of TFIIA with TBP in the TFIID complex. In this work, we have investigated the effect of TAFIIs on the interaction of TFIIA with TBP. Several different experimental approaches were used to demonstrate that TAFIIs can inhibit the binding of TBP with TFIIA and DNA. We have shown that a subset of TFIIA mutations is more defective for interaction with TFIID than with TBP, suggesting that TAFIIs can destabilize interactions between TBP and TFIIA (Figs. 1 and 2). Limited proteolysis of TFIID markedly stimulated DNA binding which suggests that degradation of TAFIIs may alleviate repression of TFIID-DNA binding activity (Figs. 4 and 5). Limited proteolysis also stimulated the amount of D-A complex formed, further suggesting that TAFIIs may also inhibit TFIIA from binding TBP. Purified human TAFII250 was found to inhibit T-A complex formation, demonstrating that at least one TAFII precludes the interaction between TFIIA and TBP (Fig. 6). Finally, we show that transcription activation by Zta can be enhanced in vivo by the inactivation of TAFII250 in ts13 cell lines by shifting to the nonpermissive temperature (Fig. 7). Taken together, these results suggest that TAFIIs can regulate transcription by inhibiting TBP interactions with TFIIA and DNA.
We have found that a subset of TFIIA mutations are more defective for
interaction with TFIID than with TBP (Figs. 1 and 2). Phenylalanine
substitution mutations at Tyr-65 and Trp-72 were capable of forming
a stable T-A complex in both Mg2+-agarose and
polyacrylamide EMSA but were defective in forming a stable EMSA complex
with TFIID (Fig. 2). These mutants were also incapable of supporting
activated transcription in vitro, indicating that TFIIA must
associate with TFIID to function in transcription (Fig. 2). Further
examination of these mutations revealed that they formed an aberrant
T-A complex, with either an increased dissociation rate (
Y65F) or
increased protease sensitivity (
W72F and W72A) (Fig. 3). Thus,
subtle changes in the stability or conformation of the T-A complex were
sufficient to disrupt association with TFIID and inhibit transcription
activation function. The TAFIIs further destabilize the
association of these TFIIA mutants with TBP, thus revealing an
inhibitory effect on TFIIA assembly. Additionally, these TFIIA mutants
may fail to induce changes in the TAFII configuration
necessary for TFIIA-TFIID-DNA complex formation to occur. In either
case, a stable interaction between TFIIA and TBP appears essential to
overcome TAFII inhibition of TFIID-DNA binding.
The potential inhibitory role of TAFIIs on TFIID binding was further supported by the stimulation of TFIID-DNA binding by limited proteolysis (Figs. 4 and 5). We found limited proteolysis stimulated D-A complex formation significantly better than T-A complex formation. Earlier work has shown that limited proteolysis could stimulate TBP-DNA binding, and this was largely a result of the degradation of the nonconserved amino-terminal domain of TBP (54, 56, 58). Moreover, the addition of TFIIA was shown to bypass the need for the proteolysis of TBP, suggesting that TFIIA stimulated a conformational change in the amino-terminal domain of TBP (56). Our results do not exclude the possibility that stimulation of DNA binding by TFIIA involves a conformational change in the TBP amino-terminal domain. Recent findings indicate that the inhibition of DNA binding by the amino-terminal domain can be relieved by interaction with the SNAP complex, which is required for activation of the U6 gene (58). The configuration of the amino-terminal domain in the TFIID complex is unknown, and it is possible that TAFIIs may increase its inhibitory effect on TBP-DNA binding.
The limited proteolysis experiments suggest that some TAFIIs in the native TFIID complex inhibit TBP-DNA binding (Figs. 4 and 5). TAFII250 has been shown by others to inhibit TBP binding to DNA (21, 28, 32). Because the addition of recombinant TAFII250 to TBP does not necessarily reconstitute the physiological properties of the entire TFIID complex, it was important to demonstrate that TAFIIs have inhibitory activities in the context of the native TFIID complex. TAFII250 was highly sensitive to proteolysis in the TFIID complex and thus qualifies as a candidate target of protease-mediated stimulation of TFIID-DNA binding (Fig. 4B). We have also shown that purified TAFII250 can inhibit T-A complex formation and that preincubation of TFIIA with TBP reverses this inhibition (Fig. 6). Because native TFIID consists of a preassembled TBP-TAFII250 complex, it is not yet clear how TFIIA overcomes TAFII250 repression in assembled TFIID. Interaction of TFIIA with other TAFs and activators may be required for TFIIA-TFIID-DNA complex formation.
Several lines of evidence indicate that TFIIA and TAFIIs interact and may be dependent upon each other for proper transcription function. TFIIA binds directly to dTAFII110 in vitro, and mutations in dTAFII110 that disrupt TFIIA binding do not function properly in transcription in vivo (59, 60). TFIIA inhibits the cross-linking of human TAFII55 and p31 to promoter sequences upstream of the TATA element and enhances the cross-linking of TAFII250 and TAFII135 to sequences downstream of the adenovirus major late promoter TATA element (40). TFIIA is required for an activator-mediated conformational change in TFIID that results in an enhanced interaction downstream of the transcriptional initiation site (20, 41). Together, these results indicate that TFIIA can bind to and alter the organization of TAFIIs in the TFIID complex. Although it is possible that TFIIA may dissociate TAFII250 from TBP, it is likely that TAFII250 remains associated with the TFIIA-TFIID-promoter complex by its association with multiple other TAFs in the TFIID complex.
The yeast homologue of TAFII250 also dissociated TBP from DNA and prevented TFIIA binding to TBP (61, 62). A partially conserved amino-terminal domain of yTAFII145 bound directly to TBP and was required for the inhibition of TBP-DNA and TBP-TFIIA binding. Interestingly, an amino-terminal deletion mutation of yTAFII145 was found to be temperature-sensitive in yeast, and this conditional lethality could be rescued by overexpression of TFIIA subunits (62). This latter result suggests that TFIIA and the amino-terminal domain of yTAFII145 function cooperatively and not antagonistically as might be predicted from the in vitro binding studies. yTAFII145 may inhibit TBP binding and TFIIA access but nevertheless stabilizes these associations once formed in vivo. These results emphasize the potential complexity of interactions between TFIID, TFIIA, and activators. Furthermore, representational display analysis of transcripts from yeast cells grown under nonpermissive conditions for yTAFII145 reveal that an equal number of transcripts were enhanced compared with the numbers that were diminished (38). This is consistent with a role of yTAFII145 in transcriptional repression as well as coactivation for some genes in yeast. Our data suggest that mammalian TAFII250 has similar activities involved in the inhibition of transcription for some activators and/or promoters in vivo. We suggest that the inhibition of TFIIA-TBP-DNA complex formation by TAFII250 may be one mechanism by which TAFII250 regulates gene expression. Activators like Zta may facilitate conformational changes and/or displacement of TAFII250 that allow TFIIA to bind TBP and promote active preinitiation complex formation.
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ACKNOWLEDGEMENT |
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We thank R. Tjian for baculovirus human TAFII250 and E. Wang for plasmids.
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FOOTNOTES |
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* This research was supported in part by American Cancer Society Grant MV-547 (to M. C.) and National Institutes of Health Grants GM54687 (to P. M. L.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a National Institutes of Health National Research Service Award and a Veterans of Foreign Wars post-doctoral cancer fellowship during this study.
Supported by United States Public Health Service National
Research Service Award GM07185.
** Also supported by the Leukemia Society of America. To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce Street, Rm. 351, Philadelphia, PA 19104. Tel.: 215-898-9491; Fax: 215-898-0663; E-mail: lieberman{at}wista.wistar.upenn.edu.
1 The abbreviations used are: TBP, TATA-binding protein; TAFII, TBP-associated factor; yTAF, yeast TAF; TFIIA and TFIID, transcription factors IIA and IID, respectively; wt, wild type; hIID, holo-TFIID; EMSA, electrophoresis mobility shift assay; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus.
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REFERENCES |
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