Vol. 12, No. 5
MOLECULAR AND CELLULAR BIOLOGY, May 1992, p. 2372-2382
0270-7306/92/052372-11$02.00/0 Copyright © 1992, American Society for Microbiology
Biochemical and Genetic Characterization of a Yeast TFIID Mutant That Alters Transcription In Vivo and DNA Binding In Vitro AND
FRED WINSTON*
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 14 January 1992/Accepted 26 February 1992
A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent sptl5 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition. The eukaryotic general transcription factor TFIID plays a central role in transcription initiation. Binding of TFIID to the TATA box of RNA polymerase II-dependent promoters is the first step in the assembly of a complex that contains RNA polymerase II and at least four other general transcription factors (TFIIA, TFIIB, TFIIE, and TFIIF) (5, 6, 60). This initiation complex is sufficient to support basal levels of transcription in vitro (11, 32, 44, 45, 49, 50). In addition, previous studies have shown that TFIID is important in vivo; in yeast cells, it is essential for growth and for normal transcription (7, 13). TFIID has been studied in vitro in some detail, and it has been shown to be capable of several interactions: binding to DNA, interactions with other general factors, and interactions with transcription regulatory factors (for a review, see reference 42). Since it is the first step in the assembly of the general initiation complex, binding of TFIID to DNA is thought to be a likely target for the regulation of transcription initiation. In support of this idea, several studies have suggested that TFIID interacts with a number of specific transcription activator proteins (21, 22, 25, 30, 51, 56). In addition, another class of transcription regulatory proteins, variously termed coactivators, mediators, or adaptors, may be required to relay a signal from some specific transcription activators to the general initiation complex via an interaction with TFIID (3, 28, 41). Finally, other results suggest that TFIID may prevent the formation of inactive chromatin over a promoter (69). These in vitro studies indicate that activators may directly facilitate binding of TFIID to the promoter to overcome repression by nucleosomes (68, 70, 71). Genes that encode TFIID have been isolated from a number of species, and a comparison of the predicted amino acid sequences shows a very high degree of conservation (80 to 90% identity) in the carboxy-terminal 180 amino acids of these proteins (for a review, see reference 42). Within this carboxy-terminal region there are two imperfect repeats of approximately 60 amino acids that are 30% identical (7, 18,
*
Corresponding author.
34, 57). Deletion analysis of yeast TFIID has demonstrated that the carboxy-terminal region is essential for growth in vivo (40) and transcription and DNA binding in vitro (24, 43). Analysis of dominant negative mutations that change amino acids in the direct repeats of TFIID and that abolish DNA binding suggested that the two repeats form a bipartite DNA binding domain (43). In contrast to the carboxy termini, the amino-terminal regions of TFIID proteins from different species show no striking similarity. For human and Drosophila melanogaster TFIID, the amino-terminal regions have been implicated as targets for the action of regulatory proteins (39, 41). In yeast strains, the requirement for this region in normal transcription remains unclear; however, strains that encode only the carboxy-terminal region of TFIID are viable (10, 14, 40, 43, 72). We have previously reported the isolation of mutations in the Saccharomyces cerevisiae SPTJ5 gene, which encodes TFIID (13). These mutations were identified as suppressors of insertion mutations in the 5' regions of the HIS4 and LYS2 genes caused by the yeast retrotransposon Ty or its solo long terminal repeat derivative (5). These insertion mutations inhibit adjacent gene expression by abolishing or otherwise altering transcription (for a review, see reference 4). Previous work showed that mutations in several SPT (suppressor of Ty) genes, including at least one mutation in SPT15, suppress these insertion mutations by restoring functional transcription (for reviews, see references 4 and 62). To begin to correlate the transcriptional changes observed in sptl5 mutants in vivo with the biochemical defects of the mutant TFIID gene products in vitro, we have used biochemical and genetic approaches to study the defects caused by one particular sptl5 mutation. Here we report that this sptl5 mutation, sptl5-122, changes an amino acid in a highly conserved region of the second repeat in TFIID. This amino acid change leads to altered DNA binding in vitro and altered transcription initiation in vivo. In addition, analysis of mutations causing other related amino acid changes strongly suggests that the highly conserved region of both repeats participates in the same aspect of a critical TFIID function, possibly DNA recognition. 2372
Downloaded from http://mcb.asm.org/ on February 3, 2016 by UNIVERSITY OF NEW SOUTH WALES
KAREN M. ARNDT, STEPHANIE L. RICUPERO, DAVID M. EISENMANN,
YEAST TFIID MUTANTS
VOL. 12, 1992
MATERIALS AND METHODS
plasmid pKA13. Plasmids for the expression of the wild-type and mutant SPT1S genes in E. coli were derivatives of the T7 RNA polymerase expression vector, pET3b (47). The 2.4-kb EcoRI-BamHI fragment from pDE32-1 (13) containing SPT15 and the 2.4-kb EcoRI-BamHI fragment from p969-1 containing sptl5-122 (L205F) were subcloned into the polylinker of M13mpl9. Oligonucleotide (oligo)-directed mutagenesis (29) (Bio-Rad Laboratories In Vitro Mutagenesis Kit) was used to introduce an NdeI restriction site at the ATG start codon of each gene. The 1,143-bp NdeI-BamHI restriction fragment from each gene was then subcloned into the NdeI-BamHI sites of pET3b to generate plasmids pKA9 (SPT15) or pKA10 (sptl5-122). To simplify the construction of plasmids for analysis of the TFIID direct repeats, a Sall restriction site was inserted 24 bp 5' to the ATG start codon of SPT15 by oligo-directed mutagenesis. Plasmids containing this Sall site and the SPT15 gene within the 2.4-kb EcoRI-BamHI fragment from the SPT15 locus fully complement sptl5 mutations. A 2.4-kb EcoRI-BamHI fragment containing the SPT15 gene and the upstream SalI site was subcloned into the EcoRI-BamHI sites of plasmid pFW4 (a YIp5 derivative with the SalI site destroyed) (38, 63) to create pKA12. To introduce specific mutations into the first repeat of SPT15, the 536-bp SallXbaI fragment was subcloned into M13mpl8 and subjected to oligo-directed mutagenesis. To introduce specific mutations into the second repeat of SPT1S, the 628-bp XbaIBamHI fragment of SPT15 was subcloned into M13mpl8 and subjected to oligo-directed mutagenesis. The presence of the desired mutation, as well as the absence of any undesired changes, was confirmed by DNA sequence analysis. Mutagenized SalI-XbaI (first repeat) or XbaI-BamHI (second repeat) restriction fragments were then subcloned into pKA12 to replace the wild-type SPT15 sequence, generating yeast-integrating plasmids containing the sptl5 mutations that encode the amino acid changes listed in Table 2. Two different null alleles of SPT15 were used in this study. Plasmid pDE51-5 contains the spt1SAlOl::LEU2 allele and was constructed by deleting the 161-bp XbaI-HindIII fragment of SPT15 in pDE25-2 (13) and replacing it with a 2.2-kb XbaI-HindIII fragment containing the S. cerevisiae LEU2 gene. Plasmid pKA23 contains the sptlSA102::LEU2 allele and was constructed by deleting the 778-bp Spel-Hindlll
fragment of pKA12 and inserting a 2.2-kb XbaI-HindIII LEU2 fragment. The deletion-insertion in pKA23 removes all but the last 15 codons of the SPT1S gene. Gap repair and sequencing. For the gapped rescue (37) of sptl5 mutations, plasmid pDE38-9 was digested with EcoRI or plasmid pDE59-1 was digested with EcoRI and BamHI to delete an -2.4-kb fragment containing the SPTI5 open reading frame. Vector fragments containing SPT15 flanking sequences were transformed into sptlS mutant strains: pDE38-9 for strains FW969 and FW971 and pDE59-1 for strains FY174, FY177, FY184, FY200, and FY259. Plasmid DNA was recovered from Ura+ or Trp+ transformants and transformed into E. coli for propagation (19). The isolation of mutant sptlS genes was confirmed by transformation of the recovered plasmids into an sptl5 strain (FW1259 or FY255). For each sptl5 mutation, both strands of the sptl5 open reading frame were sequenced by using synthetic primers. Strains, media, and Northern (RNA) hybridization analysis. Rich (YPD), minimal (SD), synthetic complete (SC), and sporulation media were prepared as described previously by Rose et al. (46). Presporulation medium (GNA) was prepared as described previously by Swanson et al. (59). The suppression of 8 insertion mutations at HIS4 and LYS2 and other nutrient auxotrophies were scored on SD media supplemented with the appropriate amino acids or SC media lacking the appropriate amino acid. Yeast transformants were selected on SC media lacking the appropriate amino acid, and ura3 strains were selected on SC media containing 5-fluoro-orotic acid (5-FOA) prepared as described previously (46). The S. cerevisiae strains used in this study are derived from S288C (A Tot gal2) and are listed in Table 1. Strains were constructed by using standard genetic methods for mating, sporulation, and tetrad analysis (46) or by one- or two-step gene replacements (48, 52). Yeast strains were transformed by the lithium acetate procedure (26). The sptlS-122 mutation was recombined into strains FY167 and FY546 by two-step gene replacements with plasmid pKA13. His' recombinants (FY474 and FY475) were verified as correct by genetic crosses with SPT15 strains, by complementation with a CEN ARS plasmid containing the SPT1S gene (pDE73-21), and by Southern hybridization analysis. Diploid strains FY548 and FY418 contain null alleles of the SPT1S gene. The 3.3-kb SpeIBamHI restriction fragment from plasmid pDE51-5 which contains the sptlSAlOl::LEU2 allele was recombined into strain FY547 by transformation and selection for Leu+ transformants. The sptl5A1O2::LEU2 allele was recombined into strain FY547 by transformation with the 2.9-kb SnaBIBamHI restriction fragment from pKA23 and selection for Leu+ transformants. Replacement of one of the wild-type SPT1S alleles in FY547 with sptlSAlOl::LEU2 or sptlSA 102::LEU2 was confirmed by tetrad analysis (2:2 for viability) and by Southern hybridization analysis. Analysis of site-directed mutations in the direct repeats of TFIID was performed as follows. Yeast-integrating plasmids (marked by URA3) containing the mutations were linearized with the restriction enzyme SpeI and transformed into strain FY548 (plasmids containing SPT15, L114F, G119V, G21OV, L205A, L205I, or L205K) or linearized with SnaBI and transformed into strain FY418 (plasmids containing SPT15+, L114F, L114A, L114I, L114K, G119P, G210P, K127A, K218A, K127R, K218R, or L205D). The plasmids were digested with SpeI or SnaBI to target integration to SPT15. Strains in which the plasmid had integrated adjacent to the sptlSA allele were identified after sporulation and
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Plasmids. Plasmids were constructed and isolated from Escherichia coli strains by using standard methods (2). DNA transformations into E. coli HB101 were performed as described previously (2). For use in DNA sequencing, plasmids were isolated by the alkaline lysis method. Isolation was followed by equilibrium sedimentation in CsCl gradients. Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs (Beverly, Mass.) and Boehringer Mannheim Biochemicals (Indianapolis, Ind.). One plasmid (pDE38-9) used for the gapped rescue of sptlS mutant alleles contains a 6.6-kb ClaI-SacI restriction fragment from the plasmid pFW213 (13) subcloned into the ClaI-SacI sites of pRS316 (CEN6 ARSH4 URA3) (54). A second plasmid (pDE59-1) used for gapped rescue contains the same ClaI-SacI fragment cloned into pRS314 (CEN6 ARSH4 TRPI) (54). The gapped rescue of the sptl5-122 gene (described later) from strain FW969 with plasmid pDE38-9 yielded the plasmid p969-1. The 6.6-kb ClaI-SacI restriction fragment from p969-1 was subcloned into the ClaI-SacI sites of the yeast-integrating plasmid pRS306 (54) to create the
2373
2374
ARNDT ET AL.
MOL. CELL. BIOL.
TABLE 1. Yeast strains Strain
Genotypea
MATa sptlS-122 his4-9178 lys2-173R2 ura3-52 ML4Ta sptl5-135 his4-9178 lys2-173R2 ura3-52 MA Ta sptlS-21 his4-9178 lys2-173R2 trpl Al ura3-52 MATa his4-9178 lys2-173R2 leu2A&l ura3-52 trplAl MATa sptlS-6 his4-9178 trplA63 leu2Al MA4Ta sptl5-8 his4-9178 trplA63 leu2AJ MATa sptlS-24 his4-9178 trplA63 leu2AJ MATa sptlS-3 his4-9178 lys2-173R2 ura3-52 trplA63 MA4Ta sptl5-21 his4-9178 lys2-173R2 leu2AO ura3-52
FY259
ALTa sptlS-7 his4-9178 lys2-173R2 leu2AJ ura3-52 trplA63 ALTa lys2-173R2 leu2Al ura3-52 trplAl A4 Ta/MA Ta sptlSAlO2::LEU2ISPTJS his4-9178/his49178 lys2-173R2/lys2-17R2 trplAl/trplAl leu2Al/ leu2Al ura3-52Iura3-52 AMTa sptlS-122 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-122 his4-9128 lys2-173R2 trplA63 ura352 AL4Ta sptl5-305::sptlSAl0l his4-9178 lys2-173R2 Ieu2Al ura3-52 trplAl MATa sptl5-301 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-303 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl AL4Ta sptlS-304 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptl5-122 lys2-173R2 ura3-52 ALTa sptlS-311::sptlSAlO2 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptl5-309 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-310 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa his4-9128 lys2-173R2 trplA63 ura3-52 AL4Ta/MA Ta his4-9178/his4-9178 Iys2-173R21lys2173R2 trplAl/trplAl leu2Al1/leu2Al ura3-52/ura3-52 AL4 Ta/MA Ta sptlSAl01::LEU2/SPT1S his4-9178/his49178 Iys2-173R2/lys2-173R2 trplAll/trplAl leu2AJ/ leu2Al ura3-52/ura3-52
trplAl
FY384 FY418 FY474 FY475 FY499
FY503 FY504 FY505
FY508 FY532 FY540 FY541 FY546 FY547
FY548
a The amino acid substitutions in TFIID encoded by the various sptl5 alleles are listed in Materials and Methods.
tetrad analysis of several transformants. These strains gave rise to haploid segregants in which the Ura+ and Leu+ phenotypes cosegregated. For each site-directed mutation, an original diploid integrant or a haploid segregant (for nonlethal mutations) in which the plasmid recombined next to the sptl5 null locus was analyzed in two ways, Southern hybridization analysis and DNA sequencing of the mutation after recloning from the genome, to verify the presence of the desired mutation (64). We found it necessary to confirm the presence of the site-directed mutation because occasionally different transformants with sptlS mutant plasmids unexpectedly gave rise to haploid segregants with a wild-type phenotype. Sequence analysis of plasmids recovered from such wild-type segregants revealed that these strains had in fact lost the desired mutation, presumably by recombination or gene conversion with the SPT1S copy in the diploid or with the adjacent sptlSAlOl::LEU2 locus. For those mutations that did not cause lethality, the phenotype conferred by the mutation was also determined in the absence of the adjacent sptlS null locus after selection on
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FW969 FW971 FW1259 FY167 FY174 FY177 FY184 FY200 FY255
5-FOA. The site-directed mutations that encode amino acid changes in TFIID are as follows: sptl5-301 for L114F, sptl5-302 for G119V, sptl5-303 for L205A, sptl5-304 for L205I, sptl5-305 for L205K, sptl5-306 for G21OV, sptlS-309 for L114A, sptl5-310 for L1141, sptlS-311 for L114K, sptl5312 for G119P, sptl5-313 for K127A, sptlS-314 for K127R, sptl5-315 for L205D, sptl5-316 for G210P, sptlS-317 for K218A, and sptl5-318 for K218R. Northern hybridization analysis was performed as described previously by Swanson et al. (59). Expression and purification of TFIID. E. coli BL21(DE3)/ pLysS (58) was transformed with plasmids pKA9 and pKA10. Transformed strains were grown at 30°C in LB broth containing 25 ,ug of ampicillin per ml and 30 ,ug of chloramphenicol per ml to an optical density at 600 nm of 0.4. IPTG (isopropyl-,-D-thiogalactopyranoside) was added to a final concentration of 0.4 mM, and incubation at 30°C was continued for 90 min. Proteins were purified by using the method of Schmidt et al. (53) as modified by Workman et al. (71) through a heparin-Sepharose CL-2B column (Pharmacia). The buffer used throughout the purification contained 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH (pH 7.9), 20% (vol/vol) glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride and the appropriate concentration of KCl. The lysis buffer and the final dialysis buffer contained 0.1 M KCl. Extracts were loaded on DEAE-cellulose (Whatman DE52) and heparin-Sepharose columns in purification buffer containing 0.1 M KCI. TFIID was eluted from the heparin-Sepharose column as described previously by Workman et al. (71). Aliquots of the final preparations were quick-frozen in liquid nitrogen, stored at -70°C, and used immediately after thawing. Samples were not refrozen, since this treatment significantly reduced the DNA binding activity of the TFIIDL205F protein. The estimated purities of the proteins were 50% for wild-type TFIID and 20% for TFIID-L205F. The relative amounts of TFIID protein in the final preparations were determined by quantitative analysis of Coomassiestained sodium dodecyl sulfate-polyacrylamide gels. We estimate the relative error in our measurements to be less than 20%. DNase I protection. An end-labelled DNA probe encoding the adenovirus major late TATA box was prepared from plasmid pRW (5). Plasmid DNA was restricted with EcoRI and HinclI. The top strand of the fragment was radiolabelled by filling in the EcoRI site with Kienow enzyme (Boehringer Mannheim) and [a-32P]dATP (Amersham). An end-labelled probe containing the S. cerevisiae his4-9128 promoter region (-387 to +97, relative to the HIS4 transcription start site) was prepared from plasmid pKAL. Plasmid pKA1 contains the 1.9-kb Sall restriction fragment of YCp701 (17) inserted at the SalI site of pUC118 (61). To radiolabel the top strand of the 484-bp his4-9128 fragment, pKA1 was digested with SpeI, treated with calf intestinal alkaline phosphatase (Boehringer Mannheim), and then phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim) and [y_32p] ATP (New England Nuclear). The phosphorylated plasmid was then digested with HaeIII. Probes were purified by electrophoresis on 5% polyacrylamide gels and electroeluted. The concentration of each probe was determined by an ethidium bromide spot test with DNA standards of known amounts. DNase I protection assays were performed essentially as described previously by Buratowski et al. (5) by using -2 ng of adenovirus major late promoter probe or 4 to 8 ng of his4-9128 probe in a 50-,ul reaction mixture. DNase I diges-
VOL. 12, 1992
YEAST TFIID MUTANTS
68
1111
:
:
:
:1
158
IZ
II
ID
R&VI :1 1 11
*
* -t127 * 11 111 1:11 1 1 * * 218
spt15-122
(L25F)
1own sptd5-122
tions were stopped by the addition of an e-qual volume of stop mix (27) to his4-9128 reaction mixtuires or an equal volume of a modified stop mix (contains 250 I,g of sonicated salmon sperm DNA per ml instead of tRNA;as the carrier) to adenovirus major late promoter reaction mi xtures. Stopped reaction mixtures were extracted once with
MOLECULAR AND CELLULAR BIOLOGY, May 1992, p. 2372-2382
0270-7306/92/052372-11$02.00/0 Copyright © 1992, American Society for Microbiology
Biochemical and Genetic Characterization of a Yeast TFIID Mutant That Alters Transcription In Vivo and DNA Binding In Vitro AND
FRED WINSTON*
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 14 January 1992/Accepted 26 February 1992
A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent sptl5 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition. The eukaryotic general transcription factor TFIID plays a central role in transcription initiation. Binding of TFIID to the TATA box of RNA polymerase II-dependent promoters is the first step in the assembly of a complex that contains RNA polymerase II and at least four other general transcription factors (TFIIA, TFIIB, TFIIE, and TFIIF) (5, 6, 60). This initiation complex is sufficient to support basal levels of transcription in vitro (11, 32, 44, 45, 49, 50). In addition, previous studies have shown that TFIID is important in vivo; in yeast cells, it is essential for growth and for normal transcription (7, 13). TFIID has been studied in vitro in some detail, and it has been shown to be capable of several interactions: binding to DNA, interactions with other general factors, and interactions with transcription regulatory factors (for a review, see reference 42). Since it is the first step in the assembly of the general initiation complex, binding of TFIID to DNA is thought to be a likely target for the regulation of transcription initiation. In support of this idea, several studies have suggested that TFIID interacts with a number of specific transcription activator proteins (21, 22, 25, 30, 51, 56). In addition, another class of transcription regulatory proteins, variously termed coactivators, mediators, or adaptors, may be required to relay a signal from some specific transcription activators to the general initiation complex via an interaction with TFIID (3, 28, 41). Finally, other results suggest that TFIID may prevent the formation of inactive chromatin over a promoter (69). These in vitro studies indicate that activators may directly facilitate binding of TFIID to the promoter to overcome repression by nucleosomes (68, 70, 71). Genes that encode TFIID have been isolated from a number of species, and a comparison of the predicted amino acid sequences shows a very high degree of conservation (80 to 90% identity) in the carboxy-terminal 180 amino acids of these proteins (for a review, see reference 42). Within this carboxy-terminal region there are two imperfect repeats of approximately 60 amino acids that are 30% identical (7, 18,
*
Corresponding author.
34, 57). Deletion analysis of yeast TFIID has demonstrated that the carboxy-terminal region is essential for growth in vivo (40) and transcription and DNA binding in vitro (24, 43). Analysis of dominant negative mutations that change amino acids in the direct repeats of TFIID and that abolish DNA binding suggested that the two repeats form a bipartite DNA binding domain (43). In contrast to the carboxy termini, the amino-terminal regions of TFIID proteins from different species show no striking similarity. For human and Drosophila melanogaster TFIID, the amino-terminal regions have been implicated as targets for the action of regulatory proteins (39, 41). In yeast strains, the requirement for this region in normal transcription remains unclear; however, strains that encode only the carboxy-terminal region of TFIID are viable (10, 14, 40, 43, 72). We have previously reported the isolation of mutations in the Saccharomyces cerevisiae SPTJ5 gene, which encodes TFIID (13). These mutations were identified as suppressors of insertion mutations in the 5' regions of the HIS4 and LYS2 genes caused by the yeast retrotransposon Ty or its solo long terminal repeat derivative (5). These insertion mutations inhibit adjacent gene expression by abolishing or otherwise altering transcription (for a review, see reference 4). Previous work showed that mutations in several SPT (suppressor of Ty) genes, including at least one mutation in SPT15, suppress these insertion mutations by restoring functional transcription (for reviews, see references 4 and 62). To begin to correlate the transcriptional changes observed in sptl5 mutants in vivo with the biochemical defects of the mutant TFIID gene products in vitro, we have used biochemical and genetic approaches to study the defects caused by one particular sptl5 mutation. Here we report that this sptl5 mutation, sptl5-122, changes an amino acid in a highly conserved region of the second repeat in TFIID. This amino acid change leads to altered DNA binding in vitro and altered transcription initiation in vivo. In addition, analysis of mutations causing other related amino acid changes strongly suggests that the highly conserved region of both repeats participates in the same aspect of a critical TFIID function, possibly DNA recognition. 2372
Downloaded from http://mcb.asm.org/ on February 3, 2016 by UNIVERSITY OF NEW SOUTH WALES
KAREN M. ARNDT, STEPHANIE L. RICUPERO, DAVID M. EISENMANN,
YEAST TFIID MUTANTS
VOL. 12, 1992
MATERIALS AND METHODS
plasmid pKA13. Plasmids for the expression of the wild-type and mutant SPT1S genes in E. coli were derivatives of the T7 RNA polymerase expression vector, pET3b (47). The 2.4-kb EcoRI-BamHI fragment from pDE32-1 (13) containing SPT15 and the 2.4-kb EcoRI-BamHI fragment from p969-1 containing sptl5-122 (L205F) were subcloned into the polylinker of M13mpl9. Oligonucleotide (oligo)-directed mutagenesis (29) (Bio-Rad Laboratories In Vitro Mutagenesis Kit) was used to introduce an NdeI restriction site at the ATG start codon of each gene. The 1,143-bp NdeI-BamHI restriction fragment from each gene was then subcloned into the NdeI-BamHI sites of pET3b to generate plasmids pKA9 (SPT15) or pKA10 (sptl5-122). To simplify the construction of plasmids for analysis of the TFIID direct repeats, a Sall restriction site was inserted 24 bp 5' to the ATG start codon of SPT15 by oligo-directed mutagenesis. Plasmids containing this Sall site and the SPT15 gene within the 2.4-kb EcoRI-BamHI fragment from the SPT15 locus fully complement sptl5 mutations. A 2.4-kb EcoRI-BamHI fragment containing the SPT15 gene and the upstream SalI site was subcloned into the EcoRI-BamHI sites of plasmid pFW4 (a YIp5 derivative with the SalI site destroyed) (38, 63) to create pKA12. To introduce specific mutations into the first repeat of SPT15, the 536-bp SallXbaI fragment was subcloned into M13mpl8 and subjected to oligo-directed mutagenesis. To introduce specific mutations into the second repeat of SPT1S, the 628-bp XbaIBamHI fragment of SPT15 was subcloned into M13mpl8 and subjected to oligo-directed mutagenesis. The presence of the desired mutation, as well as the absence of any undesired changes, was confirmed by DNA sequence analysis. Mutagenized SalI-XbaI (first repeat) or XbaI-BamHI (second repeat) restriction fragments were then subcloned into pKA12 to replace the wild-type SPT15 sequence, generating yeast-integrating plasmids containing the sptl5 mutations that encode the amino acid changes listed in Table 2. Two different null alleles of SPT15 were used in this study. Plasmid pDE51-5 contains the spt1SAlOl::LEU2 allele and was constructed by deleting the 161-bp XbaI-HindIII fragment of SPT15 in pDE25-2 (13) and replacing it with a 2.2-kb XbaI-HindIII fragment containing the S. cerevisiae LEU2 gene. Plasmid pKA23 contains the sptlSA102::LEU2 allele and was constructed by deleting the 778-bp Spel-Hindlll
fragment of pKA12 and inserting a 2.2-kb XbaI-HindIII LEU2 fragment. The deletion-insertion in pKA23 removes all but the last 15 codons of the SPT1S gene. Gap repair and sequencing. For the gapped rescue (37) of sptl5 mutations, plasmid pDE38-9 was digested with EcoRI or plasmid pDE59-1 was digested with EcoRI and BamHI to delete an -2.4-kb fragment containing the SPTI5 open reading frame. Vector fragments containing SPT15 flanking sequences were transformed into sptlS mutant strains: pDE38-9 for strains FW969 and FW971 and pDE59-1 for strains FY174, FY177, FY184, FY200, and FY259. Plasmid DNA was recovered from Ura+ or Trp+ transformants and transformed into E. coli for propagation (19). The isolation of mutant sptlS genes was confirmed by transformation of the recovered plasmids into an sptl5 strain (FW1259 or FY255). For each sptl5 mutation, both strands of the sptl5 open reading frame were sequenced by using synthetic primers. Strains, media, and Northern (RNA) hybridization analysis. Rich (YPD), minimal (SD), synthetic complete (SC), and sporulation media were prepared as described previously by Rose et al. (46). Presporulation medium (GNA) was prepared as described previously by Swanson et al. (59). The suppression of 8 insertion mutations at HIS4 and LYS2 and other nutrient auxotrophies were scored on SD media supplemented with the appropriate amino acids or SC media lacking the appropriate amino acid. Yeast transformants were selected on SC media lacking the appropriate amino acid, and ura3 strains were selected on SC media containing 5-fluoro-orotic acid (5-FOA) prepared as described previously (46). The S. cerevisiae strains used in this study are derived from S288C (A Tot gal2) and are listed in Table 1. Strains were constructed by using standard genetic methods for mating, sporulation, and tetrad analysis (46) or by one- or two-step gene replacements (48, 52). Yeast strains were transformed by the lithium acetate procedure (26). The sptlS-122 mutation was recombined into strains FY167 and FY546 by two-step gene replacements with plasmid pKA13. His' recombinants (FY474 and FY475) were verified as correct by genetic crosses with SPT15 strains, by complementation with a CEN ARS plasmid containing the SPT1S gene (pDE73-21), and by Southern hybridization analysis. Diploid strains FY548 and FY418 contain null alleles of the SPT1S gene. The 3.3-kb SpeIBamHI restriction fragment from plasmid pDE51-5 which contains the sptlSAlOl::LEU2 allele was recombined into strain FY547 by transformation and selection for Leu+ transformants. The sptl5A1O2::LEU2 allele was recombined into strain FY547 by transformation with the 2.9-kb SnaBIBamHI restriction fragment from pKA23 and selection for Leu+ transformants. Replacement of one of the wild-type SPT1S alleles in FY547 with sptlSAlOl::LEU2 or sptlSA 102::LEU2 was confirmed by tetrad analysis (2:2 for viability) and by Southern hybridization analysis. Analysis of site-directed mutations in the direct repeats of TFIID was performed as follows. Yeast-integrating plasmids (marked by URA3) containing the mutations were linearized with the restriction enzyme SpeI and transformed into strain FY548 (plasmids containing SPT15, L114F, G119V, G21OV, L205A, L205I, or L205K) or linearized with SnaBI and transformed into strain FY418 (plasmids containing SPT15+, L114F, L114A, L114I, L114K, G119P, G210P, K127A, K218A, K127R, K218R, or L205D). The plasmids were digested with SpeI or SnaBI to target integration to SPT15. Strains in which the plasmid had integrated adjacent to the sptlSA allele were identified after sporulation and
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Plasmids. Plasmids were constructed and isolated from Escherichia coli strains by using standard methods (2). DNA transformations into E. coli HB101 were performed as described previously (2). For use in DNA sequencing, plasmids were isolated by the alkaline lysis method. Isolation was followed by equilibrium sedimentation in CsCl gradients. Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs (Beverly, Mass.) and Boehringer Mannheim Biochemicals (Indianapolis, Ind.). One plasmid (pDE38-9) used for the gapped rescue of sptlS mutant alleles contains a 6.6-kb ClaI-SacI restriction fragment from the plasmid pFW213 (13) subcloned into the ClaI-SacI sites of pRS316 (CEN6 ARSH4 URA3) (54). A second plasmid (pDE59-1) used for gapped rescue contains the same ClaI-SacI fragment cloned into pRS314 (CEN6 ARSH4 TRPI) (54). The gapped rescue of the sptl5-122 gene (described later) from strain FW969 with plasmid pDE38-9 yielded the plasmid p969-1. The 6.6-kb ClaI-SacI restriction fragment from p969-1 was subcloned into the ClaI-SacI sites of the yeast-integrating plasmid pRS306 (54) to create the
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MOL. CELL. BIOL.
TABLE 1. Yeast strains Strain
Genotypea
MATa sptlS-122 his4-9178 lys2-173R2 ura3-52 ML4Ta sptl5-135 his4-9178 lys2-173R2 ura3-52 MA Ta sptlS-21 his4-9178 lys2-173R2 trpl Al ura3-52 MATa his4-9178 lys2-173R2 leu2A&l ura3-52 trplAl MATa sptlS-6 his4-9178 trplA63 leu2Al MA4Ta sptl5-8 his4-9178 trplA63 leu2AJ MATa sptlS-24 his4-9178 trplA63 leu2AJ MATa sptlS-3 his4-9178 lys2-173R2 ura3-52 trplA63 MA4Ta sptl5-21 his4-9178 lys2-173R2 leu2AO ura3-52
FY259
ALTa sptlS-7 his4-9178 lys2-173R2 leu2AJ ura3-52 trplA63 ALTa lys2-173R2 leu2Al ura3-52 trplAl A4 Ta/MA Ta sptlSAlO2::LEU2ISPTJS his4-9178/his49178 lys2-173R2/lys2-17R2 trplAl/trplAl leu2Al/ leu2Al ura3-52Iura3-52 AMTa sptlS-122 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-122 his4-9128 lys2-173R2 trplA63 ura352 AL4Ta sptl5-305::sptlSAl0l his4-9178 lys2-173R2 Ieu2Al ura3-52 trplAl MATa sptl5-301 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-303 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl AL4Ta sptlS-304 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptl5-122 lys2-173R2 ura3-52 ALTa sptlS-311::sptlSAlO2 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptl5-309 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa sptlS-310 his4-9178 lys2-173R2 leu2Al ura3-52 trplAl MATa his4-9128 lys2-173R2 trplA63 ura3-52 AL4Ta/MA Ta his4-9178/his4-9178 Iys2-173R21lys2173R2 trplAl/trplAl leu2Al1/leu2Al ura3-52/ura3-52 AL4 Ta/MA Ta sptlSAl01::LEU2/SPT1S his4-9178/his49178 Iys2-173R2/lys2-173R2 trplAll/trplAl leu2AJ/ leu2Al ura3-52/ura3-52
trplAl
FY384 FY418 FY474 FY475 FY499
FY503 FY504 FY505
FY508 FY532 FY540 FY541 FY546 FY547
FY548
a The amino acid substitutions in TFIID encoded by the various sptl5 alleles are listed in Materials and Methods.
tetrad analysis of several transformants. These strains gave rise to haploid segregants in which the Ura+ and Leu+ phenotypes cosegregated. For each site-directed mutation, an original diploid integrant or a haploid segregant (for nonlethal mutations) in which the plasmid recombined next to the sptl5 null locus was analyzed in two ways, Southern hybridization analysis and DNA sequencing of the mutation after recloning from the genome, to verify the presence of the desired mutation (64). We found it necessary to confirm the presence of the site-directed mutation because occasionally different transformants with sptlS mutant plasmids unexpectedly gave rise to haploid segregants with a wild-type phenotype. Sequence analysis of plasmids recovered from such wild-type segregants revealed that these strains had in fact lost the desired mutation, presumably by recombination or gene conversion with the SPT1S copy in the diploid or with the adjacent sptlSAlOl::LEU2 locus. For those mutations that did not cause lethality, the phenotype conferred by the mutation was also determined in the absence of the adjacent sptlS null locus after selection on
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FW969 FW971 FW1259 FY167 FY174 FY177 FY184 FY200 FY255
5-FOA. The site-directed mutations that encode amino acid changes in TFIID are as follows: sptl5-301 for L114F, sptl5-302 for G119V, sptl5-303 for L205A, sptl5-304 for L205I, sptl5-305 for L205K, sptl5-306 for G21OV, sptlS-309 for L114A, sptl5-310 for L1141, sptlS-311 for L114K, sptl5312 for G119P, sptl5-313 for K127A, sptlS-314 for K127R, sptl5-315 for L205D, sptl5-316 for G210P, sptlS-317 for K218A, and sptl5-318 for K218R. Northern hybridization analysis was performed as described previously by Swanson et al. (59). Expression and purification of TFIID. E. coli BL21(DE3)/ pLysS (58) was transformed with plasmids pKA9 and pKA10. Transformed strains were grown at 30°C in LB broth containing 25 ,ug of ampicillin per ml and 30 ,ug of chloramphenicol per ml to an optical density at 600 nm of 0.4. IPTG (isopropyl-,-D-thiogalactopyranoside) was added to a final concentration of 0.4 mM, and incubation at 30°C was continued for 90 min. Proteins were purified by using the method of Schmidt et al. (53) as modified by Workman et al. (71) through a heparin-Sepharose CL-2B column (Pharmacia). The buffer used throughout the purification contained 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH (pH 7.9), 20% (vol/vol) glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride and the appropriate concentration of KCl. The lysis buffer and the final dialysis buffer contained 0.1 M KCl. Extracts were loaded on DEAE-cellulose (Whatman DE52) and heparin-Sepharose columns in purification buffer containing 0.1 M KCI. TFIID was eluted from the heparin-Sepharose column as described previously by Workman et al. (71). Aliquots of the final preparations were quick-frozen in liquid nitrogen, stored at -70°C, and used immediately after thawing. Samples were not refrozen, since this treatment significantly reduced the DNA binding activity of the TFIIDL205F protein. The estimated purities of the proteins were 50% for wild-type TFIID and 20% for TFIID-L205F. The relative amounts of TFIID protein in the final preparations were determined by quantitative analysis of Coomassiestained sodium dodecyl sulfate-polyacrylamide gels. We estimate the relative error in our measurements to be less than 20%. DNase I protection. An end-labelled DNA probe encoding the adenovirus major late TATA box was prepared from plasmid pRW (5). Plasmid DNA was restricted with EcoRI and HinclI. The top strand of the fragment was radiolabelled by filling in the EcoRI site with Kienow enzyme (Boehringer Mannheim) and [a-32P]dATP (Amersham). An end-labelled probe containing the S. cerevisiae his4-9128 promoter region (-387 to +97, relative to the HIS4 transcription start site) was prepared from plasmid pKAL. Plasmid pKA1 contains the 1.9-kb Sall restriction fragment of YCp701 (17) inserted at the SalI site of pUC118 (61). To radiolabel the top strand of the 484-bp his4-9128 fragment, pKA1 was digested with SpeI, treated with calf intestinal alkaline phosphatase (Boehringer Mannheim), and then phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim) and [y_32p] ATP (New England Nuclear). The phosphorylated plasmid was then digested with HaeIII. Probes were purified by electrophoresis on 5% polyacrylamide gels and electroeluted. The concentration of each probe was determined by an ethidium bromide spot test with DNA standards of known amounts. DNase I protection assays were performed essentially as described previously by Buratowski et al. (5) by using -2 ng of adenovirus major late promoter probe or 4 to 8 ng of his4-9128 probe in a 50-,ul reaction mixture. DNase I diges-
VOL. 12, 1992
YEAST TFIID MUTANTS
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spt15-122
(L25F)
1own sptd5-122
tions were stopped by the addition of an e-qual volume of stop mix (27) to his4-9128 reaction mixtuires or an equal volume of a modified stop mix (contains 250 I,g of sonicated salmon sperm DNA per ml instead of tRNA;as the carrier) to adenovirus major late promoter reaction mi xtures. Stopped reaction mixtures were extracted once with
