INTRODUCTION

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Initiation of mRNA synthesis in eukaryotes depends upon the controlled and coordinated activities of a large number of proteins. RNA polymerase II and six different general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) assemble into a transcriptionally competent preinitiation complex (PIC) at the core promoter elements of class II genes (47). Among the general transcription factors, TFIID plays a central role in initiation as it binds specifically to the TATA box through its component TATA-binding protein (TBP) and nucleates PIC assembly (47). Gene-specific transcriptional regulators typically bind upstream of the TATA box and, in concert with coactivators and corepressors, modulate the level of initiation by affecting the assembly or activity of the PIC. Although any step in the initiation reaction potentially may be regulated by the gene-specific factors, a growing body of data suggests that some transcriptional activators enhance the rate of initiation by recruiting TFIID to the core promoter or by recruiting the RNA polymerase II holoenzyme to the nascent PIC (20, 54, 65).

The importance of the TFIID recruitment step in gene regulation is supported by both genetic and biochemical studies. In the yeast Saccharomyces cerevisiae, the rate-limiting association of TBP with the TATA box can be accelerated by the acidic activator Gcn4 (32), and artificial recruitment of TBP via a heterologous DNA-binding domain can bypass the requirement for upstream activators (8, 31, 75). Two independent genetic screens for TBP mutants that exhibit transcription activation defects in vivo uncovered mutants with significantly reduced affinity for DNA in vitro (3, 37). More recently, analysis of transcription initiation and PIC assembly on immobilized DNA templates in yeast nuclear extracts showed that two different acidic activators can facilitate TFIID recruitment (56). In agreement with these studies of yeast, biochemical studies performed with purified mammalian and viral transcription factors have demonstrated activator-mediated enhancement of TFIID binding to TATA elements (38, 71) in a manner that leads to a stable conformation change in the developing PIC (9). Activator function in these assays is critically dependent upon the presence of TFIIA and the TBP-associated factors (TAFs), which, together with TBP, comprise TFIID.

TFIIA has multiple roles in RNA polymerase II transcription. In agreement with its description as an RNA polymerase II general transcription factor, TFIIA is required for basal transcription in reaction mixtures containing TFIID (12, 51, 69, 76). However, TFIIA does not stimulate basal transcription when TFIID is replaced with TBP (41, 59, 69), even though TFIIA can increase the affinity of TBP for TATA elements in vitro (25, 36, 72, 77). In addition to its involvement in basal transcription, TFIIA has an important role in the regulation of transcription. Together with a number of activators and coactivators, TFIIA stimulates formation of the TFIID-TATA complex, perhaps by forming a molecular bridge between the regulatory proteins and TFIID (27, 33, 38, 61, 62, 71). Consistent with this idea, TFIIA can interact directly with certain activators (33, 51) and with a TAF (76). Moreover, crystallographic studies have shown that TFIIA binds to TBP and contacts DNA upstream of the TATA box (16, 70). Finally, a genetic screen of yeast identified a TBP mutant that is impaired in its response to acidic activators and defective for its interaction with TFIIA (66).

In addition to its coactivator function, TFIIA can stimulate transcription by counteracting the effects of certain repressors that target TFIID (20). Some of these repressors, such as Mot1/ADI, inhibit transcription by disrupting the TFIID-TATA complex (4), while others, such as Dr1-DRAP1/NC2, and HMG1, block subsequent steps in PIC assembly (14, 26, 45). TFIIA can also compete with TAFs that inhibit the DNA-binding activity of TBP, providing a possible explanation for the requirement of TFIIA in basal transcription reactions programmed with TFIID (34, 50). Interestingly, the coactivation and antirepression activities of human TFIIA have been found to reside in separable domains of the protein, confirming that TFIIA can employ both mechanisms to enhance TFIID function (40).

In S. cerevisiae, TFIIA is composed of two subunits, a 32-kDa subunit encoded by the TOA1 gene and a 13.5-kDa subunit encoded by the TOA2 gene (55). Both genes are essential for cell viability and show striking similarity to the TFIIA-encoding genes of higher eukaryotes. Several mutational studies have been performed to identify residues in TFIIA important for its various functions. Deletion mutations in TOA1 and TOA2 have identified regions of TFIIA required for subunit-subunit interaction and for TBP and DNA binding (28). A search for conditional mutations yielded temperature-sensitive mutations in TOA1 that reduce formation of a TFIIA-TBP-DNA complex and mutations in TOA2 that do not impair TFIIA function in vitro (28). Site-directed mutagenesis of the human small-subunit gene and the yeast TOA2 gene demonstrated the importance of the TBP-TFIIA interaction for activation of specific genes in vitro and in vivo (48, 49).

Despite these genetic studies, our current understanding of TFIIA derives mostly from its properties in highly purified, reconstituted transcription systems. To begin to elucidate the function and regulation of TFIIA in vivo, we have analyzed the interaction of TFIIA with a class of TBP mutants that exhibits promoter-specific defects in activated transcription in vivo (3). One of the mutants in this class, TBP-N159D, which is encoded by the spt15-341 mutation, has an amino acid substitution at a position that directly contacts the TATA box. A second mutant in this class TBP-P109A, which is encoded by the spt15-328 mutation, has a proline-to-alanine substitution within a loop connecting two of the  strands that form the DNA-binding surface of TBP (3, 29, 30). Both of these proteins are severely compromised for DNA binding in vitro (1, 3). We have found that TFIIA overexpression strongly suppresses the transcriptional defects of the TBP mutants and that purified TFIIA restores DNA binding to the one mutant that was tested. Using a genetic screen, we have identified three regions of TFIIA that are important for TBP recruitment or TBP-TATA complex stabilization in vivo. Finally, using a method for conditionally expressing TOA1, we have depleted Toa1 and demonstrated that promoters vary significantly in the degree to which they require normal levels of TFIIA for activation. These results suggest that TFIIA coactivates transcription in a gene-specific manner in vivo by facilitating or stabilizing the interaction of TBP with the TATA box.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast strains and media. Rich (YP), minimal (SD), synthetic complete (SC), 5-fluoroorotic acid, and inositol starvation media were prepared as described previously (58). YPGal and SCGal media contained 2% galactose. Solid YPGal and SCGal media also contained 1 mg of antimycin A/liter. Yeast transformants were selected on SC media lacking the appropriate nutrient.

toa1100

toa1100

Plasmids. Standard methods were used to construct plasmids, isolate DNA, and transform DNA into Escherichia coli (5). All plasmids used in yeast transformations were derived from the pRS series of shuttle vectors (10, 63). Plasmids recovered by glass bead lysis of yeast (23) were transformed into E. coli MH1 (19) cells for amplification.

Isolation of TFIIA mutants. The TOA1 and TOA2 genes were amplified from plasmids pLQ21 and pLQ23 by error-prone PCR (78). Primers that annealed to sequences on the pRS314 vector outside the polylinker were used. To generate a toa1 library, amplified fragments were digested with SalI and PstI and inserted into SalI-PstI-digested pRS314 (CEN ARS TRP1 vector). To generate a toa2 library, amplified fragments were cut with PstI and inserted into the PstI site of pRS314. Strain KY214 was transformed with the toa1 and toa2 mutant libraries, and transformants were replica plated to a series of media to test inositol auxotrophy, galactose utilization, and temperature sensitivity. To determine if the TOA1 mutations affected the four-helix bundle or -sandwich region of the protein, in vivo mapping experiments were performed (46). All TOA1 mutations that were able to suppress the mutant TBP mapped to the -sandwich coding region. DNA sequencing of this region revealed the nature of the TOA1 mutations. TOA2 mutations were identified by sequencing the entire TOA2 gene.

Saturation mutagenesis. An oligonucleotide was designed to incorporate random changes at codon 254 of TOA1 and was used in a site-directed mutagenesis reaction (43) with plasmid pLQ21 to construct a plasmid library of toa1 mutations. Sequencing the DNA of 10 randomly chosen library clones showed that the mutagenesis frequency was approximately 40%. This library was then transformed into KY214 and screened for mutations that suppress the phenotypes conferred by spt15-328. Of 900 transformants, plasmids from 15 candidates were recovered, passaged through E. coli, and retransformed into KY214. Eleven plasmids that retained suppression were sequenced in the region encompassing codon 254 of TOA1.

RNA analysis. Cells were grown at 30°C to 1 × 10⁷ to 2 × 10⁷ cells/ml in the appropriate media, and transcription of INO1, GAL1, GAL10, and CUP1 was induced as described in the figure legends (see also reference 3). RNA isolation and Northern hybridization analysis were performed by procedures described previously (3). DNA probes were derived from plasmids pJH310 (INO1) (22), pGAL1-GAL10 (EcoRI fragment 4812 [67] in pUC18), pCC69 (ACT1; a gift from F. Winston), pKA110 (GAL4) (1), pFW45 (HIS4) (74), and pLD2 (CUP1; a gift from M. Grunstein). Transcript levels were quantitated with a FUJIX BAS2000 phosphorimager with MacBAS, version 2.4, software.

Depletion of TFIIA with the MET3 promoter. To analyze gene activation upon TFIIA depletion, yeast strains YJL1471 expressing (HA)3-CDC6 and KY552 expressing UBI-lacI-TOA1 (Y), in which the fusion genes are both under the control of the MET3 promoter, were grown at 30°C in the appropriate noninducing media lacking methionine to an optical density at 600 nm (OD₆₀₀) of 0.3. To repress transcription of (HA)3-CDC6 and UBI-lacI-TOA1 (Y), L-methionine was added to a final concentration of 1 mM. To maintain cells in log-phase growth, the OD₆₀₀ was measured every hour throughout the course of the experiment and cultures were repeatedly diluted to an OD₆₀₀ of 0.3 with the appropriate media. The transcription of CDC6 and TOA1 was repressed by exposure to methionine for the times indicated in the figure legends so that similar percentages of cell survival were obtained. Subsequently, transcription of INO1, GAL1, GAL10, HIS4, and CUP1 was induced by adding the appropriate chemicals. Samples were taken for RNA preparation and Northern analysis. Percent viability was determined by spreading duplicate samples of cells from the depleted (with methionine) and nondepleted (without methionine) cultures on SC medium lacking methionine. To measure levels of the ubiquitin-LacI-Toa1 fusion protein, whole-cell extracts were prepared essentially as described previously (68). Protein extract from 0.25 OD₆₀₀ unit of cells was loaded in each lane and analyzed by immunoblotting with rabbit polyclonal antisera directed against Toa1. Fusion Toa1 protein levels were quantitated on a Power Macintosh 7100/80 computer with the public-domain National Institutes of Health Image program and normalized to ribosomal protein L3 levels. During the course of our experiments, we noticed that the kinetics of Toa1 depletion in SD and SC media differed slightly. Therefore, to allow a meaningful comparison of the effects of TFIIA depletion at different promoters, control experiments were performed to establish the time needed to deplete Toa1 levels to less than 5% of undepleted levels in both media conditions.

In vitro DNase I footprinting assays and electrophoretic mobility shift assays. A radiolabelled DNA probe containing the adenovirus major-late TATA box was prepared from plasmid pRW essentially as described previously (2). Wild-type TBP and TBP-N159D were purified from E. coli overexpression strains by standard chromatographic procedures (7, 52). The wild-type TBP used in all experiments was approximately 95% pure, and the TBP-N159D preparations used in DNase I footprinting and electrophoretic mobility shift assays were approximately 45 and 95% pure, respectively. Recombinant yeast TFIIA, which was approximately 70% pure, was a gift from Tony Imbalzano and Robert Kingston. In DNase I footprinting assays, wild-type TBP and TBP-N159D were incubated for 30 min at 30°C with <1 ng of the pRW probe in a reaction volume of 25 µl. Buffer conditions were as described previously (2). In the reaction mixtures that contained recombinant yeast TFIIA, 670 nM TFIIA was added before the incubation. This amount of TFIIA was found to be saturating for TFIIA-TBP-DNA ternary complex formation with both wild-type TBP and TBP-N159D. Samples were treated with DNase I and processed as described previously (2). In electrophoretic mobility shift assays, 15 nM recombinant yeast TBP and 2 µl of 1:100 dilutions of in vitro-translated TFIIA were incubated with the pRW probe for 30 min at 30°C under previously described conditions (13). TOA1 and TOA2 genes were transcribed in vitro with T7 RNA polymerase (Stratagene). Toa1 and Toa2 were cotranslated with a rabbit reticulocyte lysate system (Promega). [³⁵S]methionine was added to a portion of each translation reaction mixture to compare amounts of protein made. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these parallel reactions demonstrated that the electrophoretic mobility shift assay mixtures contained equivalent amounts of TFIIA (39). Electrophoretic mobility shift assays, carried out with unlabeled translation products, were analyzed on nondenaturing 4% polyacrylamide gels at room temperature (13).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Overexpression of TFIIA suppresses phenotypes caused by activation-defective TBP mutants. To investigate the importance of TFIIA in the recruitment of TBP to TATA elements in vivo, we have tested the ability of TFIIA to restore function to a class of TBP mutants that is activation and DNA binding defective (3). In our initial experiments, we asked whether overexpression of TFIIA could suppress the characteristic phenotypes, inositol auxotrophy (Ino) and slow growth on galactose-containing media (Gal), of five different TBP mutant strains in this class. High-copy-number plasmids expressing both TOA1 and TOA2 were transformed into the strains, and suppression was examined by replica plating the transformants to selective media lacking inositol or containing galactose. In addition, for experiments involving the temperature-sensitive (Ts) spt15-328 mutation, transformants were analyzed for their ability to grow at 37°C. As shown in Fig. 1A and Table 2, the Ino, Gal, and Ts phenotypes of the TBP mutant strains were significantly suppressed when both TOA1 and TOA2 were present on the 2µm plasmid. Specificity of suppression is indicated by the inability of TOA1 or TOA2 alone to reverse the mutant phenotypes (Fig. 1A) (39). Overexpression of TFIIA in these strains was confirmed by immunoblot analysis (39).

View larger version (85K): [in this window] [in a new window]   FIG. 1.   TFIIA overexpression suppresses the phenotypes conferred by TBP-P109A. (A) Yeast 2µm plasmids carrying TOA1, TOA2, or both genes were transformed into strain KY214. Transformants were replica plated to selective media to assay inositol auxotrophy, galactose utilization, or temperature sensitivity (SC). For the Ino and Ts phenotypes, plates were incubated at 30°C for 3 days. For the Gal phenotype, plates were incubated at 30°C for 4 days. (B) A yeast 2µm plasmid carrying SUA7 and a vector control were transformed into KY214 and replica plated to the indicated media. Plates were incubated at 30°C for 4 days to monitor the Ino and Ts phenotypes or for 5 days for the Gal phenotype.

Overexpression of TFIIA restores transcriptional activation in the TBP mutant strains. The inositol auxotrophy and galactose utilization defect caused by TBP-P109A and TBP-N159D correlate strongly with reduced transcriptional activation of the INO1, GAL1, and GAL10 genes (3). To determine whether TFIIA overexpression can rescue these activation defects in the TBP mutant strains, Northern analyses were performed. In wild-type strains, transcription of the INO1 gene is repressed by high levels of inositol and is dramatically induced when levels of inositol are low (Fig. 2A, lanes 3 and 4). However, in agreement with previous results (3), transcriptional activation of INO1 is severely impaired in strains that contain TBP-P109A or TBP-N159D (Fig. 2A, lanes 8 and 12). Whereas overexpression of TFIIA does not affect transcriptional activation of INO1 in strains containing wild-type TBP (Fig. 2A, lanes 1 and 2), overexpression of TFIIA in the TBP mutant strains restores activated transcription of INO1 to approximately 40 and 30% of wild-type levels for TBP-P109A and TBP-N159D, respectively (Fig. 2A, lanes 6 and 10). A similar result for the galactose-induced GAL1 and GAL10 genes was observed. For GAL1, overexpression of TFIIA in strains that contain TBP-P109A and TBP-N159D raises the activated-transcription levels to approximately 30 and 15%, respectively, of the levels observed in strains that contain wild-type TBP (Fig. 2B, lanes 6 and 10). These results reveal that suppression of the Ino and Gal phenotypes of the TBP mutant strains occurs at the transcriptional level. In addition, they demonstrate that the requirement for a transcriptional activator cannot be bypassed by an increase in TFIIA levels, as TFIIA overexpression does not lead to constitutive expression of INO1, GAL1, or GAL10. Instead, TFIIA apparently functions as a coactivator at these promoters.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   TFIIA overexpression suppresses the INO1, GAL1, and GAL10 transcriptional defects caused by TBP-P109A and TBP-N159D. (A) Northern analysis of INO1 transcription in strains that overexpress TFIIA. Yeast strains containing wild-type TBP (KY300), TBP-P109A (KY214), or TBP-N159D (KY231) were transformed with plasmid pLQ3 (2µm, TOA1 and TOA2) or pRS425 (vector control) and grown in repressing (R; 200 µM inositol) and derepressing (D; 10 µM inositol) conditions. (B) Northern analysis of GAL1 and GAL10 transcription in strains that overexpress TFIIA. The same yeast strains used in panel A were grown in noninducing (N; 2% raffinose) and inducing (I; 2% raffinose plus 5% galactose) conditions. In each case, the filter used in the upper panel was stripped and probed for ACT1 message as a normalization control.

TFIIA restores DNA binding to TBP-N159D in vitro. TBP-P109A and TBP-N159D are severely defective for binding to TATA elements in vitro, yet yeast strains that contain these mutant TBPs as the only source of TBP activity are viable (3). Therefore, in vivo, other factors most likely compensate for the extreme DNA-binding defects of these mutants. To determine whether TFIIA can restore TATA box binding to the mutant TBPs, we purified recombinant TBP-N159D and tested its ability to bind to the adenovirus major-late promoter (AdMLP) TATA box in the presence and absence of recombinant yeast TFIIA. In agreement with previous results from electrophoretic mobility shift assays (3), DNase I footprinting clearly demonstrates that, relative to wild-type TBP, TBP-N159D has greatly reduced affinity for the AdMLP TATA box. We detected no protection of the TATA box, even when the amount of TBP-N159D in the reaction mixture was 40-fold greater than the amount of wild-type TBP needed for half-maximal binding (Fig. 3A). However, in the presence of saturating amounts of yeast TFIIA, TBP-N159D protects the TATA region to levels similar to those observed with equivalent molar amounts of wild-type TBP (Fig. 3B). These results are in good agreement with an earlier study, which investigated the effects of TFIIA on a different set of TBP DNA-binding mutants (36). In conjunction with our genetic results on TFIIA overexpression, these findings demonstrate that TFIIA can overcome substantial impediments to TBP-TATA complex formation and support models in which TFIIA plays a role in TBP recruitment in vivo.

View larger version (82K): [in this window] [in a new window]   FIG. 3.   TFIIA increases the affinity of TBP-N159D for DNA in vitro. (A) DNase I footprinting assay of wild-type TBP and TBP-N159D binding to the AdMLP TATA box. Reaction mixtures for lanes 5 to 10 contained wild-type TBP, and reaction mixtures for lanes 11 to 16 contained TBP-N159D. Amounts were as follows: lanes 5 and 11, 7 nM; lanes 6 and 12, 15 nM; lanes 7 and 13, 30 nM; lanes 8 and 14, 60 nM; lanes 9 and 15, 120 nM; lanes 10 and 16, 240 nM. (B) DNase I footprinting assay of wild-type TBP and TBP-N159D binding to the AdMLP TATA box in the presence of TFIIA. Recombinant yeast TFIIA (670 nM) was added to the reaction mixtures in lanes 5 and 7 to 19. Reaction mixtures for lanes 6 to 12 contained wild-type TBP, and reaction mixtures for lanes 14 to 19 contained TBP-N159D. Amounts were as follows: lanes 7 and 14, 7 nM; lanes 8 and 15, 15 nM; lanes 9 and 16, 30 nM; lanes 10 and 17, 60 nM; lanes 6, 11, and 18, 120 nM; lanes 12 and 19, 240 nM. Lanes 1 to 3 in each panel contain the products of DNA sequencing reactions. In these assays, we did not detect a significant TFIIA-dependent increase in the affinity of wild-type TBP for DNA, presumably because conditions optimal for TBP-TATA complex formation were used (25).

Suppression of mutant TBPs by dominant mutations in TOA1 and TOA2. The strong mutant phenotypes caused by TBP-P109A and TBP-N159D allowed us to perform a genetic screen for mutations in TOA1 and TOA2 that suppress these mutant TBPs and, therefore, might facilitate the TBP-TATA interaction in vivo. TOA1 and TOA2 were mutagenized by PCR, and amplified fragments were used to construct mutant toa1 and toa2 libraries. The libraries were transformed into a strain containing TBP-P109A, and transformants were screened for suppression of the Ino, Gal, and Ts phenotypes. Since the endogenous wild-type copies of TOA1 and TOA2 were present in the strain, the suppressor mutations were expected to be dominant.

View larger version (117K): [in this window] [in a new window]   FIG. 4.   Effects of TOA1 and TOA2 mutations on TBP-TFIIA-DNA complex formation. (A) Locations of the amino acids altered by the dominant TOA1 and TOA2 mutations on the TFIIA-TBP-DNA crystal structure. Affected amino acids are highlighted in yellow for Toa1 and in red for Toa2. TBP, DNA, Toa1, and Toa2 are shown in blue, magenta, green, and white, respectively. The figure was adapted from reference 70. The position of Asp226 could not be accurately determined on this structure (70) and therefore is not indicated here. (B) Electrophoretic mobility shift assays were performed with recombinant wild-type TBP where indicated and a radiolabelled DNA probe containing the AdMLP TATA box. Prior to incubating the reaction mixture at 30°C for 30 min, recombinant yeast TFIIA (40 nM; lane 3) or in vitro-translated TFIIA (lanes 4 to 10) was added. Reaction mixtures for lanes 5 to 8 contained the indicated Toa1 mutant subunits cotranslated with wild-type Toa2, and reaction mixtures for lanes 9 and 10 contained the indicated Toa2 mutant subunits cotranslated with wild-type Toa1. Equivalent, but limiting, amounts of wild-type and mutant TFIIA were added to the reaction mixtures (see Materials and Methods). Lanes 11 and 12 contained an unprogrammed translation reaction mixture. (C) Electrophoretic mobility shift assays were performed with recombinant TBP-N159D and in vitro-translated TFIIA as described for panel B. A control reaction mixture containing TBP-N159D and recombinant TFIIA was loaded on the same gel to indicate the mobility of the TFIIA-TBP-DNA complex. The migration of this complex is indicated by an arrow.

The requirement for TFIIA in transcriptional activation is promoter specific. TBP-P109A and TBP-N159D exhibit promoter-specific defects in transcriptional activation (3). Activated transcription of certain genes, such as INO1, GAL1, and GAL10, is dramatically reduced in strains containing these TBP mutants, whereas activation of other genes, such as HIS3 and HIS4, is largely unaffected. These results suggest that transcriptional control at certain promoters is very sensitive to the stability of the TBP-TATA complex. Since TFIIA increases the affinity of TBP for DNA in vitro (25, 36, 72, 77) (see above) and since TFIIA overexpression restores activation to strains containing TBP-P109A or TBP-N159D, we reasoned that promoters may differ in the degree to which they require TFIIA for regulated transcription. Specifically, the properties of the TBP mutant strains suggested that activation of INO1, GAL1, and GAL10 may be more dependent upon the activity of TFIIA than activation of other promoters, such as HIS4. To test this hypothesis, we analyzed transcriptional activation from several highly regulated promoters in strains that contain wild-type TBP and reduced levels of TFIIA.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Depletion of TFIIA with the MET3 promoter. (A) Fusion genes comprised of UBI, lacI, and TOA1 are under the control of the MET3 promoter. The amino acid at the junction of ubiquitin and LacI is either M, I, Y, or R. (B) Three strains expressing, as their only source of Toa1, ubiquitin-LacI-Toa1 fusion proteins with the indicated junctional amino acids were streaked on plates lacking (MET) or containing methionine (+MET) and were grown at 30°C for 3 days. A strain containing a wild-type (Wt) TOA1 gene is included for comparison. (C) Correlation between levels of the ubiquitin-LacI-Toa1 fusion protein and cell viability. KY552 expressing ubiquitin-LacI-Toa1(Y) was grown to early log phase in SC (triangles) or SD (squares) media lacking methionine. Methionine was added at zero time to repress transcription of the fusion gene. Samples were taken at the indicated times and assayed for cell viability (solid symbols) and fusion protein levels (open symbols). Fusion protein levels were quantitated from immunoblots. At each of the time points, an immunoreactive band with a mobility similar to that of recombinant Toa1 was observed (not shown). The levels of this protein did not change substantially over the time course. Confirmation that this protein represents a Toa1 derivative was not made. However, the reduction in cell viability detected upon methionine addition indicates that this protein cannot substitute for wild-type Toa1.

View larger version (52K): [in this window] [in a new window]   FIG. 6.   TFIIA depletion impairs activation at INO1, GAL1, and GAL10. Northern analyses of INO1 (A) and GAL1-GAL10 (B) transcription following depletion of TFIIA are shown. Cdc6 and Toa1 depletion strains were grown to early log phase in media that lacked methionine and that was repressing (200 µM inositol for INO1) or noninducing (2% raffinose for GAL genes) for the gene of interest. Transcription of CDC6 and TOA1 was inhibited by the addition of 1 mM methionine where indicated, and the incubation was continued for times sufficient to achieve approximately equal levels of cell viability, as determined from control experiments. Subsequently, strains were transferred to derepressing media (lacking inositol for INO1 and 5% galactose for GAL1-GAL10). Times of exposure to methionine prior to gene activation were as follows for the Cdc6 and Toa1 depletion strains, respectively: 2 and 3 h for INO1 and 2 and 6 h for GAL1-GAL10. (C) Effect of GAL4 overexpression on GAL1-GAL10 induction. Strains carrying a GAL4 overexpression plasmid or control vector were grown as described for panel B except that the cultures were treated with methionine for 8 h prior to the addition of 5% galactose. Samples were taken for Northern analysis at the indicated times after gene induction. The zero-hour sample was removed prior to exposing cells to inducing conditions. RNA levels were normalized by measuring the A260 and by ethidium bromide staining of control gels to visualize rRNA.

View larger version (44K): [in this window] [in a new window]   FIG. 7.   TFIIA depletion does not impair activation of HIS4 or CUP1. (A) Northern analysis of HIS4 transcription following depletion of TFIIA. Cdc6 and Toa1 depletion strains were grown to early log phase in media that lacked methionine. Where indicated, transcription of CDC6 and TOA1 was inhibited by exposure of the cells to 1 mM methionine for 2.5 and 4 h, respectively. To induce HIS4 transcription, 10 mM 3-aminotriazole was added to each culture. Samples were taken at the indicated times prior to (zero-hour point) or following induction. (B) Northern analysis of CUP1 transcription in strains containing wild-type TBP (FY630) or the TBP mutants, TBP-P109A (KY214) and TBP-N159D (KY231). For induction, cells were treated with 100 µM CuSO4 for 30 min prior to harvesting. The filter shown in the upper panel was stripped and probed for ACT1 mRNA as a normalization control. (C) Northern analysis of CUP1 transcription following depletion of TFIIA. The experiment was conducted as described for panel A, except that cells were exposed to methionine for 2.5 and 6 h to achieve comparable levels of cell death for the Cdc6 and Toa1 depletion strains, respectively. CuSO4 (100 µM) was added to activate CUP1. For panels A and C, RNA levels were normalized by measuring the A260 and by ethidium bromide staining of control gels to visualize rRNA. In panel C, the two rRNA control gels were run for different periods of time.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

One important mechanism by which transcriptional activators stimulate gene expression is by accelerating the rate-limiting binding of TBP to the TATA box. Biochemical experiments have demonstrated a requirement for TFIIA and TAFs in communicating the activation signal to TBP. In this study, we sought to elucidate the role of TFIIA in transcriptional activation in vivo by exploiting the properties of a well-characterized class of TBP mutants (3). Three significant findings concerning the in vivo function of TFIIA have come from this work. First, we have demonstrated that TFIIA, when either overexpressed or altered by mutation, can restore function to the DNA binding- and activation-defective TBP mutants. These results strongly suggest that TFIIA facilitates the TBP-TATA interaction in vivo. Second, we have identified through a genetic screen three distinct regions on TFIIA that affect TBP recruitment or the stability of the TBP-TATA complex. Third, using a conditional expression system, we have demonstrated a promoter-specific involvement of TFIIA in transcriptional activation.

Together with the ability of TFIIA to restore DNA binding to TBP-N159D in vitro, our TFIIA overexpression results suggest that TFIIA assists in the recruitment of TBP to promoters in vivo. Although our data do not rule out an effect of TFIIA on TBP-TATA complex dissociation, a recruitment role for TFIIA is most consistent with previous biochemical studies that employed purified factors or yeast extracts (38, 56, 71). Our observation that overexpression of TFIIA causes an observable phenotype (i.e., suppression of the activation-defective TBP mutants) suggests that TFIIA may be limiting in vivo, at least at certain promoters. Interestingly, TFIIA overexpression also reverses the mutant phenotypes conferred by an unrelated TBP mutant, encoded by the spt15-21 allele, and by a mutation in the S. cerevisiae SPT3 gene (42). These mutations alter the interaction between TBP and the SAGA histone acetyltransferase complex (17). High-level expression of TFIIA most likely suppresses these mutations by assisting the TBP-TATA interaction in the context of a repressing chromatin structure. Therefore, TFIIA can lessen the effects of several types of impediments to TBP-TATA complex formation.

By searching for dominant mutations in TOA1 and TOA2 that suppress TBP-P109A, we have identified three different regions on TFIIA that may be involved in TFIID recruitment in vivo. Three of the TOA1 mutations, encoding the changes T254K, T254I, and S261R, alter residues along the TFIIA-DNA interface observed by crystallography (16, 70). Alanine scanning mutagenesis of charged residues near these positions caused a temperature-sensitive phenotype and a reduction in formation of a TFIIA-TBP-DNA complex in vitro (28). Crystallography of the TFIIA-TBP-TATA complex has revealed direct contacts between DNA and the Toa1 residues Thr252, Arg253, Lys255, Arg257, and Lys259 (16, 70). Two of these amino acids, Arg253 and Lys255, are in a position to make direct contacts with the phosphodiester backbone of the TATA box (70). The T254K and T254I substitutions, isolated in our mutant hunt, may create additional contacts between TFIIA and the TATA box. Such interactions might be especially important contributors to the overall stability of the TFIIA-TBP-DNA complex (72). Ser261 of Toa1 lies very near the basic amino acids that directly contact DNA upstream of the TATA box. Introduction of an arginine at this position might create a novel interaction between TFIIA and DNA. Two other mutations identified in our screen, encoding Toa1-Q280R and Toa2-E88K, also introduce basic amino acids in the proximity of DNA 5' to the TATA box (Fig. 4A), and may further extend the TFIIA-DNA interface. In agreement with these predictions, TFIIA derivatives containing the Toa1-T254K, Toa1-T254I, and Toa1-S261R subunits increased the formation and/or stability of TFIIA-TBP-DNA complexes in vitro (Fig. 4B and C).

In contrast to the behavior of the Ser261 and Thr254 replacements in Toa1, we identified three other TFIIA mutants, Toa1-D226V, Toa2-G30E, and Toa2-G30R, that significantly affected the TBP mutant phenotypes in vivo but did not detectably enhance TFIIA-TBP-DNA complex formation in vitro. Analysis of a Toa1 mutant protein in which residues 217 to 240 had been deleted demonstrated that this region is important for interacting with TBP (28). However, on one of the two reported TFIIA-TBP-DNA crystal structures, aspartic acid 226 is not in direct contact with TBP but rather lies in a highly acidic, solvent-exposed region (15, 16). On the other crystal structure, the positions of Asp226 and adjacent amino acids could not be determined accurately, indicating flexibility in this region (70). Our results suggest that this region may represent an important interaction site for factors that regulate TFIIA activity. Interestingly, three serine residues near Asp226, including Ser225, have been recently implicated in phosphorylation of Toa1 (64). Phosphorylation of TFIIA increases its ability to support TFIIA-TBP-TATA complex formation (64). Our genetic studies demonstrated that the Toa1-D226V mutant strongly suppressed the TBP-P109A mutant but significantly enhanced the Ino phenotype of the TBP-N159D mutant. The latter result is in excellent agreement with the proximity of Asp226 to the presumed sites of phosphorylation. If the D226V change interferes with phosphorylation of Toa1, then the activity of TFIIA in TBP recruitment will be reduced, further compromising TBP-TATA complex formation. The basis for suppression of TBP-P109A by Toa1-D226V is less easily explained by this model. By many other criteria, TBP-P109A and TBP-N159D behave similarly (3, 60). However, substitution of a critical proline residue in TBP that is important for DNA binding and that lies near a TFIIA contact point (16, 70) may significantly alter the conformation of TBP in such a way that it can be suppressed by the mutant TFIIA. This conclusion is consistent with the temperature-sensitive growth properties of TBP-P109A mutant strains.

Despite an extensive search for TOA2 mutations that suppress the TBP mutants, we identified nine mutations that change the same amino acid in the protein, Gly30. This residue lies at the tip of the four-helix bundle domain, within a turn that connects the two alpha helices of Toa2 (16, 70). A replacement of glycine at this position with either glutamic acid or arginine very likely affects the structure of the four-helix bundle domain. Previous studies demonstrated that the four-helix bundle domain is important for the coactivation properties of TFIIA in vitro (40). Our data provide strong support for this conclusion in vivo. Interestingly, alanine scanning mutagenesis of TOA2 generated only three temperature-sensitive alleles, two of which affect residues near glycine 30 (28). In agreement with the inability of the Toa2-G30E and Toa2-G30R proteins to enhance TFIIA-TBP-TATA complex formation in vitro (Fig. 4B and C), Kang et al. (28) concluded that the temperature-sensitive toa2 mutants were defective in a function other than TBP binding, TFIIA-TBP-TATA complex assembly, or subunit association.

The promoter-specific effects of the activation- and DNA binding-defective TBP mutants, coupled with the suppression of these mutants by high levels of TFIIA, suggested to us that the requirement for TFIIA may vary at different promoters. Indeed, depletion of TFIIA dramatically reduced activation of INO1, GAL1, and GAL10 but had no effect on activation of HIS4 or CUP1. These results complement those of Ozer et al. (49), who recently reported a temperature-sensitive toa2 mutation that reduced TBP binding and affected the transcription of a distinct set of genes, including some involved in cell cycle progression. A defect in GAL1 activation was also observed with this toa2 mutant; however, the magnitude of the effect, about twofold, was significantly smaller than what we observed by reducing TFIIA levels. This difference may be due to residual function of the Toa2 mutant protein under the conditions tested. This complexity, which is inherent to mutant analyses of essential genes, can be avoided by using depletion strategies such as that described here. In agreement with the results of Ozer et al. (49), we observed no effect on CUP1 activation following Toa1 depletion. Moreover, the TBP-P109A and TBP-N159D mutant proteins supported wild-type levels of CUP1 induction. These results are in apparent conflict with an earlier report of a TBP mutant, N2-1, which is unable to bind TFIIA in vitro and which causes a significant reduction in CUP1 activation (66). One explanation for this discrepancy is that N2-1 may be impaired for functions other than TFIIA binding and that these functions are critical for CUP1 activation. Interestingly, artificial recruitment of TFIIA by fusing N2-1 with Toa2 only partially rescued the GAL gene activation defect of the TBP mutant (66).

By depleting TFIIA to very low levels and then exposing the cells to conditions for gene activation, we have determined the relative need for TFIIA in the induction of various genes. Previous studies involving conditional depletion of TFIIA focused on genes that were constitutively transcribed and demonstrated a general requirement for TFIIA in maintaining the expression of these genes (28). By first activating INO1 and HIS4 and then depleting TFIIA levels, we have found a similar overall requirement for TFIIA in maintaining the transcription of these genes (39). Moreover, the rates at which these mRNAs decline following TFIIA depletion are similar. These results are in dramatic contrast to the promoter-specific effects in gene induction we have observed (Fig. 6 and 7). Together, our findings suggest that the requirement for TFIIA in the establishment of a preinitiation complex is highly variable; but once formed, the activated transcription complex remains TFIIA dependent to similar degrees at different genes. However, in the absence of more-direct biochemical support, our data can also be explained by a model in which the earliest rounds of transcription reinitiation at promoters such as INO1 are particularly dependent on TFIIA.

By regulating transcription in a gene-specific manner, TFIIA behaves similarly to several other generally acting transcription factors. In a recent genome-wide expression study, important components of the Srb-mediator, Swi-Snf, TFIID, and SAGA complexes were shown to affect only fractions of the RNA polymerase II-transcribed genes in yeast (24). What is the basis for the promoter specificity of TFIIA function? In keeping with the multiple functions of TFIIA, a number of causes are probable. The severity of the activation defect associated with certain TFIIA mutants depends significantly on the activator proteins involved (49). These mutants are also sensitive to differences in promoter structure in vivo (49). For some promoters, this effect of promoter structure most likely stems from the action of additional regulatory factors. For example, by selecting for genetic suppressors of TBP-P109A, we have implicated the Opi1 repressor and the Snf1 kinase pathway in the regulation of PIC assembly at INO1 (60). These findings suggest that TFIID recruitment may be impeded, directly or indirectly, by gene specific-repressor proteins or chromatin at INO1. The apparent lack of a TFIIA requirement at certain promoters, such as CUP1, may indicate that redundant mechanisms contribute to activation. CUP1 is a particularly interesting case because activation of this gene is unaffected by conditions that disfavor holoenzyme (35, 44) or TFIID recruitment (Fig. 7). Perhaps either mode of activation suffices for this promoter, or perhaps this promoter utilizes an entirely different mechanism of activation. Genetic studies on the RNA polymerase II general transcription factors should continue to elucidate the many and diverse strategies used for transcriptional activation in vivo.