INTRODUCTION

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Accurate and efficient transcription of eukaryotic protein-coding (class II) genes involves the concerted action of RNA polymerase II (RNAPII) and a host of accessory proteins. A subset of these proteins are known as the general transcription factors (GTFs) and include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in reference 34). The GTFs are being intensively studied with the objective of determining their respective functions during the different stages of RNAPII transcription, which include (i) formation of a preinitiation complex (PIC) on the promoter, (ii) melting of the promoter DNA, (iii) transcription initiation, (iv) clearance of RNAPII from the promoter, (v) elongation of the nascent transcript, and (vi) transcription termination.

Most promoters of class II genes contain both upstream regulatory elements and TATA elements. TATA elements, containing the consensus sequence TATAa/tAa/t, are located upstream of the mRNA start sites and are specific binding sites for the TATA-binding protein (TBP) subunit of TFIID (32, 36). For most class II promoters, formation of an active PIC is thought to occur by the initial binding of TFIID to the TATA element, in some cases accompanied by TFIIA. It is proposed that PIC formation then proceeds by either an ordered stepwise association of the remaining factors and RNAPII or by the direct recruitment of RNAPII holoenzyme (reviewed in reference 34). Upon PIC formation, the promoter DNA can be melted in an energy-dependent step, facilitating the initiation of mRNA synthesis and clearance of RNAPII from the promoter. In higher eukaryotes, transcription initiation usually occurs at a discrete start site located about 25 to 30 bp downstream of the TATA element. In contrast, transcription initiation in the yeast Saccharomyces cerevisiae frequently occurs at multiple sites within a window of 45 to 120 bp downstream of the TATA element (reviewed in references 17 and 45).

TFIIB plays an essential role in RNAPII transcription. The TFIIB polypeptide comprises a protease-sensitive N-terminal region that is highly conserved, followed by a protease-resistant C-terminal core domain that contains two imperfect direct repeats (2, 4, 31, 33). The N-terminal region contains a putative zinc-ribbon motif (W. Zhu, Q. Zeng, C. M. Colangelo, M. Lewis, M. F. Summers, and R. A. Scott, Letter, Nat. Struct. Biol. 3:122-124, 1996) and is required for interaction with TFIIF and RNAPII (3, 9, 12, 18, 35). The C-terminal core domain binds TBP (8, 18, 30) and the TBP-associated factor TAF40 (16) and interacts with DNA both immediately upstream and downstream of the TATA element (25, 26). In light of these multiple sets of interactions, TFIIB is often viewed as a bridging factor between promoter-bound TFIID and the remainder of the general transcription machinery. TFIIB may also play a role in the response to transcriptional activator proteins, as mutations in both the N-terminal and the C-terminal domains that reportedly impair activation have been identified (40, 42, 43, 47) and many transcriptional activator proteins directly bind TFIIB (10, 11, 13, 19, 28, 29, 41, 48).

In S. cerevisiae, TFIIB is encoded by the SUA7 gene. SUA7 was initially identified and characterized by Hampsey and coworkers in a suppressor analysis of respiration-deficient strains that contained an aberrant ATG translational initiation codon in the leader region of the CYC1 gene (cyc1-5000) (37). In those studies, two sua7 mutations that mapped to the N-terminal region of the protein and suppressed the respiration-deficient phenotype by conferring a downstream shift in transcription initiation at cyc1 were identified (38). Subsequent structure-function studies of yeast TFIIB have identified additional mutations in the N-terminal region of yeast TFIIB that alter start site utilization (3, 35).

To further investigate the domain of S. cerevisiae TFIIB involved in transcription start site utilization, we combined the genetic approach of Hampsey and coworkers with error-prone PCR and plasmid-shuffling to directly select for TFIIB mutants that confer downstream shifts in transcription initiation in vivo. The mutants isolated in this selection, combined with previously identified mutants altering start site utilization, define a highly conserved discrete domain within the N-terminal region of TFIIB involved in transcription start site recognition. To gain insight into the mechanism by which TFIIB affects start site utilization, a series of variant ADH1 and HIS3 promoters were constructed and analyzed in order to determine which feature of a promoter renders it sensitive to TFIIB mutations that confer downstream shifts in initiation. The results reported here demonstrate that the sensitivity of a promoter to TFIIB mutations is determined by neither the upstream regulatory elements, TATA elements, general promoter architecture, nor intrinsic strength of the start sites but rather is dictated by the identity of bases at or in the immediate vicinity of the normal start sites.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

S. cerevisiae strains and media. All S. cerevisiae strains used in this study are derivatives of S288C. Plasmid-shuffle strain FP227 (MAT ura3-52 trp163 sua71 cyc1-5000 + [p316/yIIB(URA3)]) was constructed by two-step gene replacement of the CYC1 gene in strain FP153 (MAT ura3-52 trp163 sua71 + [p316/yIIB(URA3)]) using the cyc1-5000-integrating plasmid pSF13. For the analysis of the effects of TFIIB mutations on ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions, the relevant ADH1 or HIS3 reporter plasmids (all containing URA3 as a selectable marker) were transformed into the following strains: FP251 (MAT ura3-52 trp163 his3200 lys2 sua71 + [p314/yIIB(TRP1)]); FP252 (MAT ura3-52 trp163 his3200 lys2 sua71 + [p314/V79L(TRP1)]); FP253 (MAT ura3-52 trp163 his3200 lys2 sua71 + [p314/R64G(TRP1)]); and FP254 (MAT ura3-52 trp163 his3200 lys2 sua71 + [p314/E62G(TRP1)]). Complete lineages of strains are available upon request.

Plasmids. Plasmid p314/yIIB-Mlu is a derivative of p314/yIIB, which contains the entire SUA7 promoter and coding region in the yeast-Escherichia coli shuttle vector pRS314 (CEN6 TRP1) and has been described previously (3). To generate p314/yIIB-Mlu, the megaprimer method of PCR site-directed mutagenesis was used to introduce a unique MluI restriction site into the coding region at the position corresponding to amino acids 94 and 95 with no resulting change in protein sequence (1). Integrating plasmid pSF13, containing the S. cerevisiae cyc1-5000 gene, was constructed by replacing the 385-bp XmaI-EcoRI CYC1 promoter fragment in plasmid pM142 with the corresponding fragment isolated from plasmid pM330 (5).

Construction of mutant sua7 libraries. The entire SUA7 insert in plasmid p314/yIIB-Mlu was amplified by PCR under error-prone conditions. Reaction mixtures (100 µl) contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl₂, 1 mM MnCl₂, 1 mM dCTP, 1 mM dTTP, 200 µM dATP, 200 µM dGTP, 200 ng of p314/yIIB-Mlu template, 100 pmol each of the M13 forward and reverse primers, and 5 U of Taq polymerase (Perkin-Elmer). Reactions were carried out for 30 cycles of 92°C for 1 min, 55°C for 1 min, and 72°C for 2 min. For the construction of the N-terminal mutant library, the 1.9-kb PCR product was digested with NdeI and MluI, and the approximately 300-bp fragment (encoding amino acids 1 to 94) was gel purified and cloned into the NdeI and MluI sites of p314/yIIB-Mlu. For the construction of the C-terminal mutant library, the 1.9-kb PCR product was digested with MluI and PstI, and the approximately 900-bp fragment (encoding amino acids 95 to 345) was gel purified and cloned into the MluI and PstI sites of p314/yIIB-Mlu. Approximately 25,000 primary E. coli transformants were obtained for each library. Nucleotide sequencing of 10 randomly selected clones revealed the mutation frequency to be on average one mutation per 200 bp.

Phenotypic analyses of mutant TFIIB strains. Mutant sua7 plasmids identified in the genetic selection were reintroduced into strain FP227 by transformation, and the cells were plated on CAA medium lacking uracil and tryptophan. The plates were incubated for 3 days at 30°C to select for cells containing both the mutant plasmid (TRP1-containing) and the endogenous plasmid containing the URA3 and SUA7 genes. The Ura⁺ Trp⁺ transformants were then purified once on CAA lacking Trp and uracil. To test for dominant growth phenotypes on lactate-containing medium, the strains were streaked on CAA-lactate lacking uracil and Trp, and the plates were incubated at 30°C for 9 days. To analyze the growth properties of strains containing the mutant sua7 plasmid as the sole source of TFIIB, the Ura⁺ Trp⁺ strains were streaked on 5-FOA medium and the plates were incubated at room temperature for 4 days. 5-FOA-resistant colonies were purified once by streaking on plates of CAA without Trp and incubating the plates at room temperature for 3 to 4 days. The mutant strains were tested for their relative growth rates on CAA-lactate medium without Trp (30°C for 7 to 10 days) and on rich medium at high temperature (37°C for 3 days) and low temperature (16°C for 4 days).

Mapping of in vivo mRNA start sites. Plasmids containing known TFIIB substitution mutants were introduced into strain FP153 by transformation, and the endogenous plasmid containing wild-type TFIIB was removed by plasmid shuffling on 5-FOA medium. To analyze CYC1 transcripts, cells were grown in liquid CAA-lactate medium lacking Trp and total RNA was prepared as described previously (39). To analyze ADH1 and HIS3 transcripts, total RNA was prepared from cells grown in liquid CAA-glucose medium lacking Trp. Primer extension analysis was carried out using 30 µg of total RNA under conditions previously described (39). The oligonucleotide primer for the analysis of CYC1 mRNA contained the sequence 5'-dGTCTTGAAAAGTGTAGCACC-3', corresponding to positions +53 to +34 (where +1 is the initiating ATG). The oligonucleotide primer for the analysis of ADH1 mRNA contained the sequence 5'-dGTATTCCAACTTACCGTGGGATTCG-3', corresponding to positions +63 to +39 (where +1 is the initiating ATG). To analyze transcripts from the ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions (all fused to the HIS3 coding region), strains FP251 to FP254 containing the relevant reporter plasmids were grown in liquid CAA medium without uracil and total RNA was prepared and analyzed by primer extension. The oligonucleotide primer contained the sequence 5'-dGGTTTCATTTGTAATACGC-3', corresponding to HIS3 positions +45 to +26 (where +1 is the initiating ATG).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Isolation of S. cerevisiae TFIIB mutants conferring downstream shifts in transcription initiation. To isolate TFIIB mutants conferring alterations in transcription initiation, two mutant sua7 libraries that contained substitutions in either the highly conserved N-terminal region (residues 1 to 94) or the C-terminal core domain (residues 95 to 345) were constructed. The libraries were generated utilizing PCR amplification under error-prone conditions to randomly introduce mutations into the SUA7 gene on a plasmid containing the TRP1 gene as a selectable marker (see Materials and Methods). The sua7 libraries were independently introduced into strain FP227, which contains a chromosomal deletion of the essential SUA7 gene, a plasmid containing the URA3 and SUA7 genes to maintain cell viability, and a chromosomal cyc1-5000 gene (Fig. 1). The cyc1-5000 mutation, characterized by Hampsey and coworkers, contains an aberrant ATG translational initiation codon positioned upstream and out of frame with the normal CYC1 initiation codon (21). The reduced amount of iso-1-cytochrome c in this strain results in an inability to grow on media containing nonfermentable carbon sources such as lactate. Importantly, downstream shifts in transcription initiation can generate transcripts that initiate between the aberrant and normal ATG codons, thereby partially restoring iso-1-cytochrome c protein levels and conferring the ability to grow on medium containing lactate as the sole carbon source (37). For each sua7 library, approximately 7 × 10⁵ Trp⁺ transformants of strain FP227 were plated on medium lacking tryptophan and containing lactate as the sole carbon source. After incubation at 30°C for 10 days, approximately 70 colonies were observed with the N-terminal library and approximately half that number for the C-terminal library. To determine whether growth on lactate medium was conferred by a mutant sua7 plasmid, plasmids containing the TRP1 gene and a candidate sua7 gene were rescued (53 colonies from the N-terminal library and 33 colonies from the C-terminal library) and reintroduced into strain FP227, and the transformants were again tested for growth on CAA/lactate-Trp. For the N-terminal library, 32 of the 53 rescued plasmids conferred the ability to grow on CAA-lactate medium without Trp when retested, whereas 0 of 33 rescued plasmids from the C-terminal library conferred the ability to grow on CAA-lactate without Trp. These results suggest that the C-terminal core domain of TFIIB does not play a significant role in the mechanism of start site utilization.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Scheme used for the genetic selection of TFIIB mutants that confer downstream shifts in transcription initiation in vivo.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   TFIIB mutations confer downstream shifts in transcription initiation at the CYC1 and ADH1 promoters. Total RNA (30 µg) from yeast strains containing the indicated TFIIB mutations were analyzed by primer extension utilizing a CYC1-specific (A) or ADH1-specific (B) oligonucleotide primer. The numbers to the left of each panel indicate the major transcription start sites and the distance from the translation-initiating ATG (+1).

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Differential effect of the V79L TFIIB mutation on transcription initiation at the ADH1 and HIS3 promoters. Primer extension analyses were performed using an ADH1-specific (A) or HIS3-specific (B) primer as described in the legend to Fig. 2. The first four lanes in each panel contain sequencing ladders of each promoter region (left to right: G, A, T, C) obtained with the same primers used in the primer extension analyses.

The ADH1 promoter contains a single functional TATA element and both TFIIB-sensitive and TFIIB-insensitive major transcription start sites. The ADH1 promoter contains three major transcription start sites. In our strains these sites map to positions 37, 28, and 27 (where +1 corresponds to the translation-initiating ATG) and an additional minor start site maps to position 38. Although the TFIIB mutants clearly confer decreased utilization of the 38 and 37 sites along with more pronounced utilization of minor start sites further downstream, the utilization of the 28 and 27 sites appears relatively constant in both wild-type and mutant TFIIB strains (Fig. 2B and 3A). One possible explanation for this result is that the ADH1 promoter contains two functional TATA elements, one that directs initiation to the 38 and 37 sites and a more downstream TATA that directs initiation to the 28 and 27 sites. The relatively constant amount of the 28 and 27 transcripts could be explained by TFIIB mutations causing downstream shifts for initiation directed by both the upstream and downstream TATA elements. In this view, a downstream shift in initiation directed by the more downstream TATA element would give rise to the observed far-downstream transcripts as well as a reduction in the amount of the 28 and 27 transcripts. However, a downstream shift for initiation directed by the more upstream TATA element would give rise to a reduction in the amount of the 38 and 37 transcripts along with an increase in the amount of the more downstream 28 and 27 transcripts, thereby restoring essentially wild-type levels of the 28 and 27 transcripts. Alternatively, the ADH1 promoter could contain a single functional TATA element and the constant level of the 28 and 27 transcripts might be due to these sites being insensitive to the effects of the TFIIB mutations. To distinguish between these possibilities, we sought to identify the functional TATA element(s) in the ADH1 promoter. Examination of the sequence upstream of the 38 start site revealed two potential TATA elements located between positions 128 and 123 (TATAAA) and between positions 110 and  105 (TATTAA) (Fig. 4A). Site-directed mutagenesis was utilized to disrupt each of these motifs with the substitution of an XbaI restriction site (Fig. 4A). To facilitate primer extension analysis of the variant ADH1 promoters in strains containing a wild-type chromosomal ADH1 gene, plasmids that contained the wild-type or mutant ADH1 promoters fused to the HIS3 coding region were constructed. The plasmids were then introduced into wild-type or mutant TFIIB strains that contained a deletion of the chromosomal HIS3 gene. Primer extension analysis of total RNA using a HIS3-specific primer revealed that substitutions at positions 127 and 124 (D1) abolished all detectable ADH1 transcription whereas substitutions at positions 109, 107, and 106 (D2) had no detectable effect (Fig. 4B). These results demonstrate that the ADH1 promoter contains a single functional TATA element located between positions 128 and 123 and suggest that the 38 and 37 start sites and the 28 and 27 start sites differ in their intrinsic sensitivity to the effects of TFIIB mutations.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   The ADH1 promoter contains a single functional TATA element. (A) Sequence of the ADH1 promoter region from positions 137 to 98 (where +1 corresponds to the initiating ATG) and specific mutations introduced to disrupt the two potential TATA elements (D1, D2). (B) Primer extension analyses were performed using an ADH1-specific primer and 30 µg of total RNA isolated from yeast strains containing the indicated ADH1 promoter construct and either wild-type or mutant (V79L) TFIIB. The last four lanes contain the sequencing ladder of the ADH1 promoter obtained with the same primer used in the primer extension analysis (left to right; G, A, T, C).

Analysis of ADH1-HIS3 hybrid promoters and ADH1 promoters with altered distance between the TATA element and the start sites. Mutations in TFIIB can cause downstream shifts in transcription initiation at the ADH1 promoter, whereas no significant shifts are detected at the HIS3 promoter (Fig. 3B; data not shown) (3, 5). To gain insight into the mechanism by which TFIIB mutants alter transcription initiation, we sought to determine which features of the ADH1 and HIS3 promoters render them susceptible and insensitive, respectively, to downstream shifts. We initially tested whether alterations in the spacing between the ADH1 TATA element and the start sites result in altered sensitivity to TFIIB mutations. A series of variant ADH1 promoters containing insertions or deletions of 12, 28, or 40 bp between the TATA element and the start sites were constructed and introduced into both wild-type and V79L mutant TFIIB strains (Fig. 5). Primer extension analysis demonstrated that deletion of 12 or 28 bp or insertion of 12, 28, or 40 bp between the TATA element and the initiation region had no significant effect on the initiation pattern in the wild-type TFIIB strain or on the downstream shifts observed for the mutant strain (Fig. 5). Deletion of 40 bp resulted in a significant reduction in the amount of the upstream 38 and 37 transcripts in the wild-type TFIIB strain, but downstream transcripts were still observed in the V79L strain (Fig. 5). The reduction in the amount of the 38 and 37 transcripts in the 40-bp deletion construct was not unexpected since the 38 and 37 sites in this construct are positioned 50 bp downstream of the TATA element, close to the minimal distance required between a yeast TATA element and an initiation site. Nonetheless, these results suggest that the spacing between the ADH1 TATA element and the initiation region does not play a significant role in determining the sensitivity of the start sites to TFIIB mutations.

View larger version (53K): [in this window] [in a new window]   FIG. 5.   Analysis of ADH1 promoters with altered distances between the TATA element and the start sites. Primer extension analyses were performed with variants of the ADH1 promoter containing insertions or deletions of 12, 28, or 40 bp. The 40 and +40 constructs contain a deletion and duplication, respectively, of the region between positions 92 and 52. The 28 and +28 constructs contain a deletion and duplication, respectively, of the region between positions 92 and 64. The 12 and +12 constructs contain a deletion and duplication, respectively, of the region between positions 92 and 80.

View larger version (43K): [in this window] [in a new window]   FIG. 6.   The differential effect of TFIIB mutations on the ADH1 and HIS3 promoters is determined by the promoter regions containing the respective transcription start sites. (A) Schematic representation of the ADH1 and HIS3 promoter regions and the reciprocal HIS3-ADH1 and ADH1-HIS3 hybrid promoters. The numbers indicate the distances from the initiating ATG, and the arrows indicate the positions of the major transcription start sites. (B) Primer extension analyses were performed using a HIS3-specific primer and 30 µg of total RNA isolated from yeast strains containing the ADH1 or HIS3-ADH1 promoter constructs and either wild-type TFIIB or the indicated TFIIB mutant. (C) Primer extension analyses were performed as described in the legend to panel B using yeast strains containing the HIS3 or ADH1-HIS3 promoter constructs and either wild-type TFIIB or the indicated TFIIB mutant.

Base substitutions in the vicinity of transcription start sites can alter their sensitivity to TFIIB mutations. The preceding results strongly suggest that the DNA sequence surrounding a transcription start site determines whether that site is utilized to a lesser extent in mutant TFIIB strains with a concomitant increase in the usage of more-downstream sites. In light of these results, we addressed whether base substitutions in the vicinity of a start site could alter the sensitivity of that site to downstream shifts in mutant TFIIB strains. As noted earlier, the ADH1 38 and 37 start sites and the ADH1 28 and 27 start sites differ in their sensitivity to TFIIB mutations, with the 38 and 37 sites being significantly less utilized than the 28 and 27 sites in mutant TFIIB strains. Comparison of the sequences encompassing the 38 and 37 start sites and the 28 and 27 start sites revealed that all four start sites reside within an identical 5-bp CAAGC motif (Fig. 7A). In contrast to the C that is immediately upstream of the motif containing the 28 and 27 sites (position 30), a T is present at the homologous position upstream of the motif containing the 38 and 37 sites (position 40) (Fig. 7A). To determine whether this base difference contributes to the differential sensitivity of these sites to TFIIB mutations, a mutant ADH1 promoter containing a T-to-C substitution at position 40 was constructed and analyzed. In a wild-type TFIIB strain, the T(40)C mutant promoter yielded a slightly higher amount of the 38 and 37 transcripts than the normal ADH1 promoter and reduced levels of the 28 and 27 and further downstream minor transcripts (Fig. 7B, lanes 1 and 5). In mutant TFIIB strains, the normal ADH1 promoter displayed the classical downstream initiation pattern, yielding significantly reduced levels of the 38 and 37 transcripts relative to the 28 and 27 transcripts accompanied by increased levels of the more downstream transcripts (Fig. 7B, lanes 2 to 4). In contrast, the T(40)C promoter yielded nearly equal levels of the 38 and 37 transcripts and the 28 and 27 transcripts and significantly reduced levels of the more-downstream transcripts (Fig. 7B, lanes 6 to 8). These results suggest that the T(40)C substitution increases utilization of the 38 and 37 sites in a wild-type TFIIB strain and renders these sites less sensitive to downstream shifts in mutant TFIIB strains.

View larger version (50K): [in this window] [in a new window]   FIG. 7.   Base substitutions in the vicinity of transcription start sites can alter their sensitivity to TFIIB mutations. (A) Presented is the DNA sequence of the transcription initiation regions in the ADH1 and HIS3 promoters. Underlined in bold is the CAPuGC motif present at both of the ADH1 start sites and at the HIS3 23 start site. (B) Primer extension analyses were performed as described in the legend to Fig. 6 using yeast strains containing the ADH1 or T(40)C mutant ADH1 promoter constructs and either wild-type TFIIB or the indicated TFIIB mutant. (C) Primer extension analyses were performed using yeast strains containing the ADH1-HIS3 or G(25)T mutant ADH1-HIS3 promoter constructs and either wild-type TFIIB or the indicated TFIIB mutant.

View larger version (45K): [in this window] [in a new window]   FIG. 8.   The sensitivity of transcription start sites to TFIIB mutations is determined by the sequence in the immediate vicinity of the start sites and not by their intrinsic strength. Primer extension analyses were carried out as described in the legend to Fig. 6 using yeast strains containing the ADH1 or the indicated variant ADH1 promoter construct and either wild-type TFIIB (A) or the V79L TFIIB mutant (B). (C) Quantitation of 38 and 37 start site utilization. Phosphorimaging and Molecular Analyst software were used to quantitate ADH1 transcripts from three independent primer extension analyses. Utilization of the 38 and 37 sites was plotted as the percentage of major ADH1 transcripts (38 plus 37 divided by the sum of 38, 37, 28, and 27). The sensitivity of the 38 and 37 sites to the effects of the V79L TFIIB mutation was defined by the sensitivity index (S.I. = utilization of the 38 and 37 sites in the wild-type TFIIB strain divided by that in the V79L strain).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we investigated the domain and residues of S. cerevisiae TFIIB involved in the utilization of transcription start sites and the underlying reason for why TFIIB mutations cause downstream shifts in transcription initiation at some but not other promoters. To identify residues of TFIIB involved in the utilization of start sites, we modified the genetic approach of Hampsey and coworkers to directly select for TFIIB mutants conferring downstream shifts in transcription initiation in vivo (37). This approach resulted in the isolation of 24 sua7 alleles that conferred downstream shifts in transcription initiation at the CYC1 and ADH1 promoters in vivo. The mutants contained single or multiple amino acid substitutions in the highly conserved N-terminal region of the TFIIB protein and in all cases involved a substitution for Glu-62, Arg-64, Arg-78, or Val-79. Seventeen of the mutants, all with substitutions for Arg-78 or Val-79, conferred a dominant phenotype for the ability to grow on lactate in the presence of wild-type TFIIB. The remaining seven mutants, all containing substitutions for Glu-62 or Arg-64, displayed a recessive phenotype for growth on lactate medium. All of the mutants also exhibited a cold-sensitive phenotype.

It is noteworthy that among the 15 mutants with multiple amino acid substitutions, 5 of them contained substitutions of Tyr-30. Single-substitution mutants of Tyr-30 were not identified in the selection, but rather, these substitutions were always found in combination with a substitution of Arg-78 or Val-79. We think it unlikely that these Tyr-30 substitutions are fortuitous for several reasons. First, three different substitutions for this residue were identified. Second, the Tyr-30 substitutions were always in combination with an Arg-78 or Val-79 substitution. Third, in two cases the Tyr-30/Arg-78 double-mutant strain grew better than the corresponding Arg-78 mutant strain. Further work will be required to determine the role of Tyr-30 in the function of both wild-type TFIIB and the Arg-78 and Val-79 mutant proteins.

In previous studies utilizing site-directed mutagenesis, we also reported substitutions of residues Trp-63 and Phe-66 that conferred downstream shifts in transcription initiation. Thus, substitutions of residues Glu-62, Trp-63, Arg-64, Phe-66, Arg-78, or Val-79 have been identified that confer downstream shifts in transcription initiation, thereby defining a highly conserved discrete domain within the N-terminal region of TFIIB involved in the utilization of start sites. This domain is positioned adjacent and C terminal to a zinc-ribbon motif in TFIIB that is required for stable RNAPII-TFIIB binding (35). Continued biochemical analyses are needed to determine the primary biochemical alteration associated with the mutant TFIIB proteins.

As observed previously, TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters but not at the HIS3 promoter (Fig. 3). This differential effect of TFIIB mutations on these promoters could be due to differences in (i) the upstream regulatory regions, (ii) the TATA elements, (iii) the spacing between the TATA elements and the start sites, (iv) specific sequences between the TATA elements and the start sites, and/or (v) specific sequences in the immediate vicinity of the start sites. To shed light on the mechanism by which TFIIB influences the utilization of start sites, we sought to exploit the promoter-specific effect of the TFIIB mutations and determine which of the above promoter features renders the ADH1 and HIS3 promoters sensitive and insensitive, respectively, to TFIIB mutations. We initially examined whether the distance between the TATA element and the start sites plays a role in establishing the sensitivity of those sites to TFIIB mutations. Analysis of a series of ADH1 promoters containing insertions or deletions between the TATA element and the initiation region demonstrated that alteration in the spacing of this region had no significant effect on the sensitivity of this promoter to TFIIB mutations (Fig. 5). A sequence-dependent basis for the differential sensitivity to TFIIB mutations was initially suggested by the observation that the ADH1 38 and 37 start sites and the ADH1 28 and 27 start sites, separated by only 10 bp, differ in their sensitivity to TFIIB mutations (Fig. 4). Results from the analysis of reciprocal HIS3-ADH1 and ADH1-HIS3 hybrid promoters supported this view, demonstrating that the differential sensitivity of the HIS3 and ADH1 promoters to TFIIB mutations was not due to differences in promoter architecture or sequences far upstream of the start sites, but rather was due to differences in the specific sequences in the immediate vicinity of the respective start sites (Fig. 6). Examination of the sequence of the ADH1 and HIS3 start sites revealed that the ADH1 38 and 37 sites and the ADH1 28 and 27 sites reside within a CAAGC motif while the HIS3 23 site is contained within the sequence CAGGC, both sharing homology with a PyAA/TPu proposed consensus sequence for S. cerevisiae RNAPII start sites (14, 20, 22). The suggestion that the differential sensitivity of these start sites to TFIIB mutations is due to sequence differences in the immediate vicinity of the sites was further supported by the demonstration that a T(40)C substitution conferred decreased sensitivity of the ADH1 38 and 37 sites to TFIIB mutations, whereas a G(25)T substitution rendered the HIS3 23 start site more sensitive (Fig. 7).

The preceding results demonstrated that base substitutions in the immediate vicinity of start sites could alter their sensitivity to TFIIB mutations. However, since the ADH1 T(40)C substitution rendered the 38 and 37 start sites less sensitive to downstream shifts in mutant TFIIB strains but also modestly increased their utilization in a wild-type TFIIB strain, it was unclear whether the sensitivity of start sites to TFIIB mutations was dictated by specific sequences or simply by the intrinsic strength of the start site. The analysis of 16 additional variant ADH1 promoters containing single-base substitutions upstream, downstream, or in the position of the 38 and 37 start sites demonstrated that sensitivity was not correlated with overall strength, but rather was dictated by the identity of bases at or in the immediate vicinity of the start sites (Fig. 8). This was evidenced by (i) the T(42)C substitution, which conferred the highest utilization of the 38 and 37 sites in a wild-type TFIIB strain yet the same degree of sensitivity seen for the normal ADH1 promoter (S.I. = 3.0) in the V79L mutant TFIIB strain; (ii) the T(40)A, T(40)C, and T( 40)G substitutions, which conferred little or no change in 38 and 37 utilization in the wild-type strain but decreased sensitivity (S.I. = 1.7 to 2.1) in the V79L strain; (iii) the A(38)T substitution, which conferred no change in 38 and 37 usage in the wild-type strain yet dramatically increased sensitivity in the V79L strain (S.I. = 15); and (iv) the A(38)G substitution, which abolished utilization of the 38 site in the wild-type strain yet surprisingly supported efficient utilization in the V79L strain. The last result also provides the first example of a start site sequence that is utilized preferentially in a mutant TFIIB strain, suggesting that TFIIB mutations may not only impair normal start site recognition by RNAPII but also confer altered specificity as well.

As noted earlier, transcription initiation in higher eukaryotes usually occurs at a discrete site located about 30 bp downstream of the TATA element, whereas transcription initiation in S. cerevisiae often occurs at multiple sites within a window of 45 to 120 bp downstream of the TATA element. The ability of S. cerevisiae RNAPII to initiate within an extended window downstream of the TATA element reflects a fundamental difference in the initiation mechanism compared to that in higher eukaryotic cells, and the basis for this difference is unknown. Thus, it remains of significant interest to determine both the mechanism by which S. cerevisiae RNAPII initiates transcription at such a distance from the TATA element and the manner in which wild-type and mutant forms of TFIIB affect the utilization of potential start sites. One possibility to account for the difference in the mammalian and yeast initiation patterns is that the positioning of yeast PICs on a promoter is fundamentally different from that of mammalian PICs. Yeast PICs could potentially adopt a much more elongated conformation than mammalian PICs and/or loop out intervening DNA in order to contact downstream start sites. Although this remains a possibility, it has been reported that promoter melting for the S. cerevisiae GAL1 and GAL10 genes in vivo is comparable to that seen in other eukaryotes, occurring approximately 20 bp downstream of the TATA element (15). Moreover, the extent of melting was reported to be independent of the distance between the TATA element and the transcription start sites. Thus, the positioning and promoter melting of yeast PICs appears to be similar to that of mammalian PICs. In light of these results, Giardina and Lis proposed that S. cerevisiae RNAPII may be released from the PIC and scan downstream DNA for preferred start sites (15).

Results from both genetic and biochemical studies strongly suggest that RNAPII and TFIIB are solely responsible for determining the start site of transcription in S. cerevisiae. Genetic studies have shown that mutations in the largest subunit of RNAPII (Rpb1) can confer downstream shifts in transcription initiation that are similar to those observed with TFIIB mutations (6). In contrast, deletion of the Rpb9 subunit of RNAPII, or mutation disrupting a C-terminal zinc-ribbon in Rpb9, confers an upstream shift in transcription initiation (23, 46). Biochemical studies by Kornberg and colleagues have shown that the pairwise exchange of S. cerevisiae TFIIB and RNAPII with their homologs from Schizosaccharomyces pombe is sufficient to convert the transcription initiation pattern to that of S. pombe (27). Thus, an attractive extension to the scanning model is that a TFIIB-RNAPII interaction governs the start site recognition properties of RNAPII and that TFIIB mutants confer downstream shifts in transcription initiation by impairing start site recognition. Initially proposed by Hampsey and colleagues, such impairment could cause RNAPII to bypass sites normally used for initiation and allow for initiation at sites further downstream (38). Our results here demonstrate that the sequence at or in the immediate vicinity of a start site determines the tendency of that site to be underutilized in mutant TFIIB strains accompanied by increased usage of further downstream start sites. These results are consistent with the view that a TFIIB-RNAPII functional interaction, either direct or through the action of another factor, affects the efficiency by which potential start sites are recognized and utilized by a scanning polymerase. The incorporation of TFIIB mutants into additional genetic, biochemical, and molecular studies should prove useful in determining the precise mechanism by which S. cerevisiae RNAPII recognizes and utilizes potential transcription start sites.