Cell, Vol. 65. X33-340, April 19, 1991, Copyright 0 1991 by Cell Press
A Highly Conserved Domain of TFIID Displays Species Specificity In Vivo Grace Gill and Robert Tjian Howard Hughes Medical Institute and Department of Molecular and Cell Biology University of California Berkeley, California 94720
Summary Recombinant TFIID proteins from yeast, Drosophila, and human function interchangeably In vitro to restore basal level transcription to a human HeLa extract depleted for TFIID. Here we report that the recently cloned human and Drosophila TFIID genes fail to substitute in vivo for the S. cerevlsiae TFIID gene, SPTIL, which is essential for viability. Analysis of yeast-human hybrid TFIID proteins reveals that the failure of human TFIID to functionally replace yeast TFIID maps to the highly conserved C-terminal domain. Thus, the C-terminal consenred domain of TFIID, as well as the N-terminal divergent domain, appears to be involved in species-specific interactions. Introduction The factors required for proper initiation of transcription by RNA polymerase II can be roughly divided into two classes. One class, the general transcription factors, ap pears to be required for accurate initiation at all promoters, and the other class, the promoter-specific factors, modulates the level of transcription at only certain promoters. Many of the mechanisms by which specific factors alter the rate of transcription initiation are thought to be highly conserved from yeast to mammals. For example, a number of mammalian transcription factors, such as the glucocorticoid and estrogen receptors, activate transcription when introduced into the yeast Saccharomyces cerevisiae (Metzger et al., 1988; Schena and Yamamoto, 1988). Conversely, the yeast activator GAL4 stimulates transcription from promoters bearing GALebinding sites in mammalian, Drosophila, and plant cells (Fischer et al., 1988; Kakidani and Ptashne, 1988; Ma et al., 1988; Webster et al., 1988). Mutations that increase the activity of one activating region of GAL4 have similar phenotypes in yeast and mammalian cells, supporting the idea that the mode of action of at least some transcription factors has been conserved from yeast to mammals (Gill et al., 1990). Recent biochemical studies suggest that components of the general transcription machinery are also highly conserved among eukaryotes. The sequences of the large subunit of RNA polymerase II from yeast and human, for example, are very similar (Allison et al., 1985; Sweetser et al., 1987). There are at least five general factors, TFIIA, -B, -D, -E, and -F, required in addition to RNA polymerase II for accurate and efficient initiation in the HeLa in vitro transcription system (Buratowski et al., 1989; Reinberg et al., 1987; Reinberg and Roeder, 1987). Fractions isolated
from S. cerevisiae will substitute for at least two of the HeLageneral factors, TFIIA and TFIID, in vitro(Buratowski et al., 1988; Cavallini et al., 1988; Hahn et al., 1989; Horikoshi et al., 19896). In fact, yeast TFIID was initially identified as an activity from yeast that restores basal transcription to a TFIID-depleted HeLa extract. Similarly, the HeLa TFIID fraction restores activity to a reconstituted yeast in vitro transcription system depleted of TFIID (Flanagan et al., 1990). The recent cloning of TFIID genes from a variety of organisms (Cavallini et al., 1989; Fikes et al., 1990; Gasch et al., 1990; Hoey et al., 1990; Hoffman et al., 1990a, 1990b; Horikoshi et al., 1989a; Kao et al., 1990; Muhich et al., 1990; Peterson et al., 1990; Schmidt et al., 1989) provides an opportunity to test whether the conservation of TFIID function observed in vitro applies in vivo. The binding of TFIID to the TATA box is thought to be the first step in the assembly of an active transcription complex at the promoter (Buratowski et al., 1989; Van Dyke et al., 1988). Comparison of the predicted protein sequences of TFIID from a variety of organisms reveals a highly conserved C-terminal domain of 180 amino acids and an N-terminal domain that varies in both length and sequence. Truncated proteins bearing only the conserved C-terminal domain from yeast, Drosophila, and human TFIID are able to bind specifically to the TATA box and interact with the other general factors, such as TFIIA and TFIIB, in order to restore basal level transcription to a TFIID-depleted HeLa nuclear extract (Hoey et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). The integrity of this highly conserved C-terminal domain appears to be essential for function since even small deletions in this region of yeast TFIID disrupt both DNA binding and transcriptional activity in vitro (Horikoshi et al., 1990). In addition to its crucial role in basal transcription, TFIID has been proposed to be the target, direct or indirect, of several promoter-specific activators (reviewed in Lewin, 1990; Ptashne and Gann, 1990). For example, an affinity column of the strong acidic activator VP18 has been shown to bind both human and yeast TFIID (Stringer et al., 1990). None of the recombinant TFIID proteins, however, restores stimulation by upstream factors, such as Spl, USF, or GAL4-VP18, to a reconstituted HeLa transcription system (Hoffman et al., 1990b; Peterson et al., 1990; Pugh and Tjian, 1990; F. Pugh and R. Tjian, unpublished data). Thus, a novel class of transcription factors, termed coactivators, which are thought to copurify with TFIID, has been proposed to mediate the productive interaction of promoter-specific activators with the basal transcription machinery. Although the variable N-terminal domain of TFIID is dispensable for basal level transcription in vitro, the N-terminal domains of Drosophila and human TFIID appear to be required for a productive interaction with the Spl coactivator (Peterson et al., 1990; Pugh and Tjian, 1990). Genetic studies have demonstrated that the S. cerevisiae TFIID gene, SPT75, is essential for viability (Eisenmann et al., 1989). Yeast bearing weak alleles of SPTIS, although viable, have pleiotropic phenotypes, including
altered transcription initiation and slow growth. We have used a complementation assay in S. cerevisiae to begin mapping functional domains of TFIID in vivo. TFIID from Schizosaccharomyces pombe, whose sequence is 93% identical in the C-terminal domain to that of S. cerevisiae, complements a null mutation in SPT75 (Fikes et al., 1990). Since the amino acid sequences of human and Drosophila TFIID are greater than 80% identical to S. CerevisiaeTFllD in the conserved region and since yeast TFIID will largely substitute for human TFIID in vitro, we tested the ability of both human and Drosophila TFIID to functionally substitute for yeast TFIID in vivo. In addition, we have tested the ability of several yeast-human chimeric TFIID genes to complement a disruption of the essential yeast TFIID gene. Our findings suggest that a highly conserved region of this essential protein exhibits species-specific functional differences.
Transform yeas1 test strain with LEU2 plasmid expressing TFIID.
Results The ability of the human and DrosophilaTFIIDstofunctionally substitute for the yeast TFIID (yTFIID) in vivo was tested using the plasmid shuffle technique (Boeke et al., 1987; see Figure 1). The endogenous TFIID gene of the test strain (yt6A15) was disrupted by replacing the 5’ end of the gene with the TRP 7 gene. Because the yTFllD gene is essential for viability, our test strain carries the wild-type yTFllD gene on a single-copy URA3 plasmid. Heterologous TFIID genes, expressed from the yeast ADH promoter, were introduced intothisstrain on asecond plasmid bearing the LEU2 selectable marker. The LEU’ transformants were then replica plated to media containing 5fluoroorotic acid (5FOA). Only those cells that have lost the URA3 plasmid, carrying yTFIID, will grow on 5-FOA plates. Thus, only if the TFIID allele on the LEUP plasmid functionally substitutes for yTFllD will the strain grow on the 5FOA plates. As shown in Figure 2, the test strain (yt6A15) transformed with a LEUP plasmid lacking TFIID sequences fails to grow on 5-FOA plates, confirming that the yTFllD on the URA3 plasmid is essential for viability. When the wild-type yTFllD gene is carried on the LEU2 plasmid, the LEU’ transformants are viable on 5-FOA plates. In contrast, when LEU2 plasmids expressing either the full-length human (hTFIID) or Drosophila (dTFIID) TFIID are introduced into the test strain, the transformants fail to grow on 5-FOA plates, although they grow normally on LEU- plates. This indicates that the human and Drosophila homologs fail to functionally substitute for yTFIID. It is interesting to note that although human and Drosophila TFllDs might be expected to compete with yTFllD for binding to the TATA box or other general transcription factors, we do not observe any growth defect of strains expressing both yeast and human or Drosophila TFIID. lmmunoblot analysis (see below and Figure 5) confirms that each of the heterologous TFllDs examined here is indeed expressed in yeast. Since the N-terminal divergent region of both the human and DrosophilaTFllD proteins is longer, contains a stretch of polyglutamine, and lacks the high density of charged amino acids found in the N-terminus of yTFllD (as schema-
Kcplica-plate to 5-I’(),\ plates lo select for cells that have lost the llRA.3 plasmid.
1. The Plasmid Shuffle Assay for Function in S. cerevisiae
The endogenous yeast TFIID gene (SPT75) of the test strain was disrupted by replacing the 5’ end of the gene, including amino acids l-61, with the TRP7 gene. This yeast strain (yt6A15) carries the wildtype yTFllD gene on a single-copy URA3 plasmid (pRS316yllD). Heterologous TFllDs were introduced into this strain on a multicopy plasmid bearing the LEUZ gene as a selectable marker. The LEU’ transformants were then replica plated to media containing 5-FOA to select against the URA3 plasmid carrying the wild-type yTFIID. Thus, the strain is only viable on 5-FOA plates if the TFIID allele on the LEUP plasmid functionally substitutes for yTFIID.
tized in Figure 6) one possible explanation for the lack of complementation by the human and Drosophila homologs is the presence or absence of species-specific N-terminal sequences. To address this possibility, truncated proteins bearing only the conserved C-terminal domain were tested for their ability to substitute for yTFIID. Deletion of amino acids 7-57 of yTFllD does not disrupt DNA binding or transcription in vitro (Horikoshi et al., 1990). As shown in Figure 2, yeast bearing only a truncated yTFll0 (A2-57)
Specificity of TFIID In Vivo
Figure 2. Ability of Various TFIID Genes to Complement a yTFll0 Disruption (A) Full-length and truncated yeast, human,
and Drosophila TFIIDs. (B) Yeast-human hyyTFIID
brid TFIIDs. The yeast-human hybrid TFllDs are diagrammecl in Figure 3. Multicopy plasmids carrying the L/X/2 selectable marker and the indicated TFIID allele expressed from the yeast ADH promoter were introduced into the test yeast strain (yt6Al5) described in Figure 1. LEU’ transformants were patched onto plates lacking leucine (LEU-) and then replica plated to both 5-FOA plates and LEU- plates. Plates were incubated at 3WC for 2 days (LEU-) or 3 days (5-FOA). These TFIID genes were also tested on single-copy plasmids with, in each case, the same results (data not shown).
consisting of the C-terminal 183 amino acids, y183C, are viable, although they grow slowly. Truncated proteins bearing only the C-terminal 180 amino acids of hTFllD (h180C) or 191 amino acids of dTFllD (d191C) have also been shown to function in DNA binding and transcription invitro(Hoeyet al., 1990; Petersonet al., 1990). Incontrast to the results with y183C, when plasmids expressing hl8OC or d191C are introduced into yt6A15, the Leu+ transformants fail to grow on 5-FOA plates. Thus, it appears that the failure of the human and Drosophila homologs to substitute for yTFllD maps to the C-terminal domains, which are >80% identical. We then examined several yeast-human hybrid TFllDs in the plasmid shuffle assay to localize the functional difference between human and yeast TFIID. Each of the hybrids diagrammed in Figure 3 is designated by a three-letter code indicating the species origin (yeast or human) of each of three regions used to generate the hybrids: the divergent N-terminal region (yTFIID residues 1-61; hTFllD residues 1-159); the first 110 amino acids of the conserved region; and the remainder of the C-terminal region (69-70 amino acids). Yeast bearing a disruption of yTFllD and transformed with a hybrid, yhh, bearing the N-terminus of yeast TFIID (amino acids i-61) and the C-terminus of human TFIID (amino acids 160-339) are not viable on 5FOA plates, whereas a hybrid protein, hyy, bearing the N-terminus of hTFllD (residues 1-159) and the C-terminus of yTFllD (residues 62-240) fully complements a disruption of SPTIS. These results support the conclusion that the functional difference between yeast and human TFIID lies in the C-terminal domain. Surprisingly, both the yhy and yyh hybrids, in which only part of the C-terminal region of yTFllD has been replaced with sequences from hTFIID, allow the test strain to grow on 5-FOA plates (Figure 2 and summarized in Figure a),
suggesting that the failure of hTFllD to function in yeast is due to the cumulative effect of several unfavorable amino acid substitutions in the conserved region. When a plasmid expressing the yhy hybrid is introduced into the test strain, however, the 5-FOA-resistant colonies are few and slow to appear, whereas cells bearing the yyh hybrid (yTFIID amino acids 1-171 plus hTFllD amino acids 270339) grow nearly as well as wild-type yTFIID. The differential growth of cells bearing these hybrids suggests that differences between human and yeast TFIID in the region corresponding to yTFll0 residues 62-171 are particularly unfavorable for full activity in yeast. Consistent with this view, the hhy hybrid (hTFIID residues l-269 plus yTFllD residues 172-240) fails to substitute for yTFIID, but the hyh hybrid allows growth of the test strain in the presence of 5-FOA. Thus, of the three regions tested in our hybrids, only amino acids 62-171 of yTFllD are sufficient to allow hTFllD to function in yeast. This highly conserved 110 amino acid region must therefore contain the major determinants that functionally distinguish yeast and human TFIID. Although the N-terminal 57 amino acids of S. cerevisiae TFIID are dispensable for viability, we found that the yeast strain yt6 bearing only yl83C grows slowly under all conditions
particularly poorly on a nonfermentable carbon source. This finding suggests that the N-terminal region of yTFll0 is important for proper expression of one or more genes required for growth tional yeast-human
in glycerol. Strains bearing the funchybrid TFllDs show no differential
growth in the presence or absence of a fermentable carbon source (Figure 4). In particular, the hyy hybrid bearing the human N-terminus and the yeast C-terminus not only substitutes for yTFllD but allows yeast to grow well even in the absence of a fermentable carbon source. The obser-
y TFIID yl83C I-
Figure 4. Yeast Searing Nonfermentable Carbon
IFigure 3. Schematic Diagram of the Yeast, Human, man Hybrid TFIID Proteins Analyzed in This Study
++ and Yeast-Hu-
A “++” indicates that the TFIID fully complements a disruption of yTFIID. A “f” indicates that the allele is sufficient for viability, but yeast bearing this allele grow slowly. A “-‘I indicates that the allele is not able to substitute for yTFIID. yTFllD sequences are designated by open boxes, hTFllD sequences by shaded boxes. Each hybrid is designated by a three-letter code indicating the species origin of each of the three regions composing the hybrids. Arrowheads above yTFllD and hTFllD mark the positions of restriction sites used to construct the yeasthuman hybrids. A unique Mrol site was introduced at the junction of the divergent N-terminal and COnSSNSd C-terminal domains, position 1590f hTFllD and position 61 of yTFIID. There isan Xbal siteat position 171 of yTFIID, and an Xbal site was introduced at an analogous position, residue 269, of hTFIID. The amino acids immediately flanking the junctions were not altered in the yeast-human hybrids diagrammed here. The two arrows below yTFllD indicate an imperfect direct repeat present in all the TFIID alleles shown here.
vation that a growth defect caused by deleting the N-terminus of yTFllD is overcome by substituting the unrelated human N-terminus raises the possibility that these diverged sequences may share a common function either to interact with and stabilize the C-terminus or to interact with other transcription factors. The expression of heterologous proteins in yeast was verified by immunoblot analysis using polyclonal antisera raised against human, Drosophila, or yeast TFIID. These antisera fail to react strongly with the conserved domain of TFIID and are therefore effectively species specific. As shown in Figure 5, antiserum raised against the Drosophila protein detects dTFllD expression in yeast and antiserum raised against the human protein detects hTFIID, hhy, hyy, and hyh, all expressed at comparable levels. The failure of the anti-yTFIID polyclonal antiserum to react strongly with the y183C protein allows us to examine the levels of the yeast-human hybrids yhh, yyh, and yhy in a yeast strain in which yTFllD function is provided by y183C. Although the yhh and yyh hybrids are expressed at a lower steady-state level than yTFllD in this strain, the nonfunc-
a Truncated Source
Gene Grow Poorly
The indicated TFIID allele carried on a multicopy LEU.2 plasmid is the only source of TFIID in the strain. Cells were patched onto rich medium (YepAde + 2% glucose) and then replica plated to YepAde + Gly (3% glycerol + 0.1% glucose) and YepAde + Glu (2% glucose). Plates were incubated at 30% for 24 hr. Yeast bearing both ylS3C and yTFllD genes also grow poorly on a nonfermentable carbon source (data not shown).
tional yhh hybrid is expressed at a comparable level to yyh, which is fully functional in yeast. Because functional and nonfunctional proteins are expressed at similar levels, the failure of heterologous TFIID proteins to function in yeast cannot be attributed simply to low expression or poor stability. Discussion We have found that the human and Drosophila TFIID genes fail to complement a disruption of the yeast TFIID gene in vivo. Unexpectedly, the functional difference between these proteins maps to a region whose amino acid sequence is over 80% identical. Analysis of yeast-human hybrids suggests that the failure of hTFllD to function in yeast is a cumulative effect of amino acid substitutions located throughout the conserved region. Substitutions in one region, however, particularly interfere with hTFllD function in yeast since, of the three regions tested, only yeast sequences from position 62-171 are sufficient to allow hTFllD to function in yeast. Thus, one or more of the amino acid substitutions in a 110 amino acid region of hTFllD that is 83% identical to the yeast sequence apparently disrupt an essential interaction in yeast. Of the 19 amino acid differences between yeast and human TFIID in this region, 14 of these positions also vary between yeast and Drosophila. Since the S. pombe TFIID complements a disruption of yTFllD (Fikes et al., 1990) it is tempting to speculate that the important functional difference resides at one or more of 10 positions in this region at which the S. cerevisiae and S. pombe sequences are identical to each other and different from both human and Drosophila sequences (see Figure 6).
Figure 5. lmmunoblot gous TFllDs Expressed
4 3Es,,, Yk”ha%
Analysis of Heteroloin Yeast
(A) lmmunoblots containing whole-cell extracts from yeast (yt6A15) transformed with either the control LfU2 plasmid or a plasmid expressing dTFllD were probed with polyclonal rabbit antiserum directed against dTFIID; 0.6 OD, units were loaded per lane. (6) lmmunoblotscontaining whole-cell extracts from yeast (yt6A15) transformed with either the control LEU2 plasmid or plasmids expressing hTFllD or the human-yeast hybrids hyy, hhy. or hyh were probed with rabbit polyclonal antiserum raised against hTFIID; 0.2 ODaa units were loaded per lane. (C) lmmunoblots containingwhole-cell extracts from yeast bearing y166C (yt6A15 pRS516y166C) transformed with either the control LEU2 plasmid or plasmids expressing full-length yTFll0 or the yeasuhuman hybrids yhh, yyh, or yhy were probed with rabbit polyclonal antiserum raised against yTFIID; 1 .O ODaD units were loaded per lane. This polyclonal antiserum fails to react strongly with the consented domain of TFIID, so we were unable to detect the yl66C protein, which is present in all the lanes. The arrows indicate the positions of the TFIID proteins.
6. A Comparison
(A) Schematic comparison of full-length TFIID from different species. The C-terminal conserved region is represented as an open box. Arrows represent an imperfect direct repeat present in each of the C-terminal domains. ‘0” represents an uninterrupted stretch of glutamine residues. Tandem arrowheads represent copies of an imperfect tripeptide repeat, Pro-Met-Thr. (6) Amino acid sequence comparison of the region containing the major determinants of a species-specific functional difference. The sequence of S. cerevisiae TFIID residues 62-171, a region that is sufficient to allow hTFllD to function in yeast, is presented on the top line in one-letter code. The homologous sequences of S. pombe, human, and Drosophila TFIID are presented below; a dash indicates identity to the S. cerevisiae sequence and differences are indicated in one-letter code. Note that the numbering of homologous positions differs due to the differing lengths of the N-terminal regions.
Several interesting motifs, in particular an imperfect direct repeat, have been noted in the conserved region of TFllD (Hoeijmakers, 1990; Nagai, 1990). Deletion derivatives of yTFllD containing only one complete unit of the repeat are nonfunctional in both DNA-binding and transcription assays in vitro (Horikoshi et al., 1990). Since most of the amino acid differences implicated as having a major role in determining a functional difference between yeast and human TFIID lie in the first unit of the direct repeat, our data raise the possibility that the two units of the repeat may be functionally nonequivalent in vivo. The failure of the human and Drosophila TFIID proteins to substitute for yeast TFIID is surprising not only because the sequence of these proteins is highly conserved, but also because yeast, human, and Drosophila TFIID proteins are functionally interchangeable for basal transcription in a HeLa extract in vitro, and a fraction containing HeLa TFIID restores transcription to a reconstituted yeast extract in vitro (Buratowski et al., 1988; Cavallini et al., 1988; Flanagan et al., 1990; Hoey et al., 1990; Horikoshi et al., 1988). One simple explanation for this apparent discrepancy is that the in vitro and in vivo conditions are clearly different. Viability is a crude but stringent assay; if even one essential gene fails to be transcribed the strain will not be viable. TFIID is thought to participate in at least three types of interactions to support high levels of transcription: protein-DNA interactions that result in specific binding to the TATA box, protein-protein interactions with other general factors required to assemble an active initiation complex, and protein-protein interactions that mediate upstream factor response. The functionally significant amino acid differences between yeast and human TFIID presumably form part of a surface involved in one of these types of essential interactions. Thus, there are at least threesimple explanations for the molecular nature of the difference between the yeast and human proteins. First, hTFllD may differ in its DNA recognition properties and fail to bind the promoter region of an essential yeast gene. When assayed on a large number of mutant TATA sequences in a HeLa extract in vitro, however, yeast and human TFllDs showed almost identical relative transcription activities (Wobbe and Struhl, 1990). Second, it remains possible that hTFllD fails to interact effectively with one of the yeast general transcription factors in vivo, despite the fact that the HeLa TFIID fraction restores transcription to a partially fractionated yeast nuclear extract (Flanagan et al., 1990). This implies that general transcription factors other than TFIID will also display species-specific functional differences. Third, hTFllD may fail to respond to one or more upstream regulatory factors required for expression of sufficient levels of essential genes in yeast. Although the N-terminus of hTFllD is required to mediate stimulation by certain upstream activators such as Spl (Peterson et al., 1990; Pugh and Tjian, 1990), this finding does not exclude the possibility that the conserved domain of TFIID may also be involved in mediating the response to some activators. In fact, transcription complexes containing the truncated yeast TFIID described here, y183C, respond to the
upstream activator GAL4 in vivo (G. G., unpublished data), suggesting that the conserved C-terminal domain of yTFllD must contain the residues required to mediate the activity of at least certain activators. Yeast genetics in combination with in vitro studies of DNA binding, protein-protein complex formation, and stimulated transcription may allow us to define precisely the molecular nature of the difference between the yeast and human TFIID proteins. In particular, if the human and Drosophila TFIID proteins fail to make specific proteinprotein contacts with essential yeast transcription factors, it may be possible to identify compensating mutations in those factors. As mentioned in the introduction, a new class of transcription factors has been proposed to mediate the productive interaction of the basal transcription machinery with at least some promoter-specific activators (Berger et al., 1990; Kelleher et al., 1990; Peterson et al., 1990; Pugh and Tjian, 1990). Recombinant TFIID proteins show speciesspecific differences in their response to upstream factors in vitro, suggesting that the interaction of the coactivator with TFIID is species specific. For example, Drosophila TFIID responds to stimulation by Spl in the presence of a Drosophila fraction containing the Spl coactivator but not in the presence of a human Spl coactivator fraction (Pugh and Tjian, 1990). The species specificity of the coactivator-mediated response has been attributed to the highly divergent N-terminal domain of TFIID because this region is required for an upstream factor response in the homologous system, and truncated proteins bearing only the C-terminal domain show no significant difference in their abilities to restore basal transcription in vitro. The results presented here indicate that there are also speciesspecific functional differences in the conserved C-terminal domain of TFIID. It is possible, therefore, that some of the species-specific differences observed in vitro are not due exclusively to differences in the N-terminal domains. Experimental
Yeast Strains and Media The test strain yt6A15 was constructed as follows. pRS316yllD, a single-copy MA3 plasmid expressing yTFllD off the ADH promoter, was introduced into the haploid strain yt6 (a ~~~3-52 /eu2-3,7 12 his3200ade2-701 adel lys2-807 trpl-907 arol metga14-542galSO-538). The SPT75 gene present in plasmid pDE33-1 (Eisenmann et al., 1969) was disrupted in vitro by replacing the Bglll fragment encompassing the Vend of SPT15 with a Bglll fragment carrying the TRW gene. An EcoRI-BamHI fragment containing the sptlBTRP7 disruption was then used to transform the yeast strain yt6 bearing pRS316yllD to TRP+. All TRP’transformants were shown also to be URA’. The disruption of the chromosomal SPT15 gene was verified by Southern analysis. The yeast strain yt6A15 pRS316yl63C was constructed in the same way except the MA3 plasmid used in the first transformation was pRS316y163C. All yeast transformations were performed using the LiAc procedure (Ito et al., 1963). Yeast were grown on YepAde (1% yeast extract, 2% bacto-peptone, 0.072% adenine) with either 2% glucose or 3% glycerol + 0.1% glucose as carbon sources or defined media (0.67% yeast nitrogen base without amino acids, supplemented with the appropriate amino acids and 2% glucose). 5-FOA plates, lacking leucine, were as described (Boeke et al., 1967). Plasmid Constructions The LEUP plasmid vector was pAAH5,
a 21r. plasmid
ADH promoter and terminator (Ammerer, 1963). All TFIID alleles de scribed here were cloned into the filled-in Hindlll site of pAAH for expression from the ADH promoter in yeast. pRS316yllD was mntructed as follows: An Asp716 fragment containing the yTFllD coding sequence was excised from wyTFllD (a gift from K. Kilomanski), filled in, and introduced into pAAH5. A BamHl fragment encompassing the pADH-yTFIID expression cassette was subcloned into pRS316. a single-copy URA3 plasmid (Sikorski and Hieter, 1969). A blunt-ended Ndel-Xhol fragment encoding y163C was similarfy introduced into pAAH5. A BamHl fragment containing the pADH-yl63C expression cassette was subcloned into pRS316 to generate pRS316y163C. Ndel fragments encoding dTFllD and d191C were excised from pARdTFllD and pARdTFIID-191C (Hoey et al., 1990), filled in with Klenow, and introduced into pAAH5. hTFllD and h16OC were excised as Ndel frag ments from the vaccinia expression vector (Peterson et al., 1990). filled in with Klenow, and introduced into pAAH5. The yeast-human hybrids were also cloned into the filled-in Hindlll site of pAAH for expression in yeast. Site-Dlrected Mutegenesis pBSKhllD. containing an EcoRl fragment of hTFllD cloned into the pBluescript SK+ polylinker (Stratagene), was a kind gift of Naoko Tanese. TheAsp716fragmentmntainingyTFllD(seeabove)wasfilled in with Klenow and cloned into the Hincll site of pBluescript SK+. Uracil-substituted single-stranded DNA generated from these clones was used for site-directed mutagenesis (Kunkel, 1965). The oligonucleotide 5’-TGATGTGGCGGACATATGTlllTCAGAlTC-3’ was used to introduce a unique Ndel site and a new methionine at position 57 of yTFIID, generating yl63C. The oligonucleotide 5cGCGGTACAAlTCCGGAACTCTCCGS’was used to introduce a unique Mrol site at position 159 of hTFIID, and a unique Mrol site was introduced at an analogous position of yTFIID, residue 61, with the oligonucleotide 5”GlTGGAACAAlTCCGGATGTGGCGG-3’. Smal-Mrol fragments were then exchanged to generate the hyy and yhh fusions. There is an Xbal site at position 171 of yTFIID. Theoligonuclsotide S’AAGGCCTI’CTAGACGTATAGGAAACT-3’ was used to introduce an Xbal site at an analogous position, residue 269, of hTFIID. Sequences C-terminal to the Xbal site were then exchanged between yTFllD and hTFIID, generating the hybrids yyh and hhy. The yhy hybrid was generated by replacing yhh sequences C-terminal to the Xbal site with those from yTFIID. The hyh hybrid was generated by replacing hyy sequences C-terminal to the Xbal site with those from hTFIID. Note that the introduction of Mrol and Xbal sites does not alter the amino acid sequence of the region.
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