SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo

Cdl, vol. 58, im-119l, September22, lass, Copyriiht Q

IS89 by Cell Press

SP7Y5, the Gene Encoding the Yeast TATA Binding Factor TFIID, Is Required for Normal Transcription Initiation In Vivo David M. Eisenmann, Catherine Doilard, and Fred Winston Department of Genetics Harvard Medical School Boston, Massachusetts 02115

Summary Mutations in the S. cerevisiae Sf7Y5 gene welrr isolated as suppressors of insertion mutations that siter the transcription of adjacent genes. Molecular and genetic analysis of the cloned NW5 gene has shown that it is the same as the gene that encodes the TATA binding factor TFIID. Analysis of spf75 mutants has demonstrated that alterations in TFIID can change transcrlptlon inltiation In vlvo. in additlon, we demonstrate that TFIID is essential for growth and that spfl5 mutations are pieiotropic, as spfld mutants grow slowly and have defects in both mating and sporulation. Therefore, TFIID is an essential transcription factor in vivo, likely to be required for normal expression of a large number of genes. Introduction Transcription initiation by RNA polymerase II in eukaryotes is a complex process that requires a large number of proteins in order to occur in a specific and regulated way. The identification of the components required for this event has thus far come primarily from biochemical experiments. In vitro systems derived from HeLa cells have allowed the identification of several factors, in addition to RNA polymerase II, that are required for specific initiation (Matsui et al., 1980; Samuels et al., 1982; Dignam et al., 1983; Sawadogo and Roeder, 1985). These factors, plus RNA polymerase II, form a complex at the promoter that is necessary for specific initiation (Fire et al., 1984; Hawley and Roeder, 1985, 1987; Burton et al., 1988; Reinberg and Roeder, 1987; Reinberg et al., 1987; Zheng et al., 1987; Buratowski et al., 1989). For many promoters, the initial event in the in vitro assembly of the RNA polymerase II initiation complex is the binding of the transcription factor TFIID to the TATA region (Davison et al., 1983; Sawadogo and Roeder, 1985; Van Dyke et al., 1988; Buratowski et al., 1989). The binding of TFIID is required for the subsequent assembly of the transcription factors TFIIA, TFIIB, TFIIE, and RNA polymerase (Buratowski et al., 1989) and for in vitro transcription of several different genes (Nakajima et al., 1988). Experiments of Workman and Roeder (1987) have suggested that the binding of TFIID can be inhibited by the presence of nucleosomes over the TATA region, suggesting that the state of the chromatin in the promoter region can influence transcription initiation by its effect on TFIID binding. Therefore, the ability of TFIID to bind to DNA appears to

play a central role in the initiation of transcription by RNA polymerase II. Several experiments have demonstrated a striking conservation of transcriptional mechanisms between yeast and mammalian cells, both for regulatory proteins and for components of the basic transcriptional machinery (for examples, see Allison et al., 1985; Corden et al., 1985; Kakidani and Ptashne, 1988; Lech et al., 1988; Webster et al., 1988; Struhl, 1988; Schena and Yamamoto, 1988; Chodosh et al., 1988). The yeast TFIID protein has recently been identified and demonstrated to be functionally equivalent to the mammalian TFIID protein in a HeLa in vitro transcription system (Buratowski et al., 1988; Cavallini et al., 1988). These results suggest that studies of RNA polymerase II initiation in yeast will reveal fundamental aspects of transcription initiation that will prove to be consewed in most eukaryotes. We have taken a genetic approach to the study of transcription initiation in the yeast S. cerevisiae. We have isolated a large number of transcription mutants as suppressors of Ty and solo 6 insertion mutations that inhibit normal transcription of adjacent genes (Winston et al., 1984a, 1987; Fassler and Winston, 1988). (8 sequences are the long terminal repeats of Ty elements [Cameron et al., 19793). Mutations in SPT genes (SPT = suppressor of Ty) restore expression of genes adjacent to these Ty and solo 8 insertion mutations by restoration of functional transcripts (Silverman and Fink, 1984; Winston et al., 1984b; Clark-Adams and Winston, 1987; Fassler and Winston, 1988; Clark-Adams et al., 1988; Hirschman et al., 1988). spr mutations have identified a large number of genes, including histone genes (Clark-Adams et al., 1988) that are required for normal transcription initiation in yeast. In addition, several SPT genes are essential for growth (ClarkAdams and Winston, 1987; C. Dollard, E. A. Malone, M. S. Swanson, and F. Winston, unpublished data). In theory, selection for spt mutants might identify transcription factors that have already been identified biochemically, as well as previously unidentified factors necessary for transcription. In this paper, we present results that demonstrate that the SPTl5 gene is the same as the gene that encodes the TATA binding factor TFIID, as cloned by Hahn et al. (see accompanying paper). Furthermore, we show that this gene is essential for growth and that leaky (nonlethal) mutations in SPT75 confer several mutant phenotypes, including altered transcription initiation. These results demonstrate that TFIID is an essential transcription factor, required in vivo as well as in vitro. Results SP775 Encodes the TATA Binding Factor TFIID We have isolated a large number of suppressors of Ty or solo 6 insertion mutations in the 5’ regions of the yeast HIS4 and LYS2 genes (Winston et al., 1984a, 1987; Fassler and Winston, 1988). The 33% gene was identified by

Cell 1194

SPT16 FUNCTION ppwzl3

+ pDE13-1

I pDEl9-1

pDE34-3

+

lkb Figure 1. Restriction

Map of the SPT75 Locus

The top line represents the SfTl5 locus and its restriction sites. The dotted portion of this line is part of the vector and the black box represents the position of the open reading frame that encodes TFIID, based on the sequence of Hahn et al. (1999a). The lines below represent the subcloned fragments. These subclones were tested for SfT15 function after transformation into the so115 mutant strain FW1259.

three mutations in one of the four complementation groups that were isolated as suppressors of the S insertion mutation, his4-9776 (see Discussion in Winston et al., 1967). Strains that contained spf75 mutations were quite sick, which suggested that they were defective in some important or essential function. To study SPTl5 in greater detail, we cloned the gene by complementation of an spt75 mutation. Strains that contain the insertion mutations his4-9176 and lys2-173R2 and that are otherwise wild-type have a His- Lys+ phenotype. The phenotypes of both of these insertion mutations are reversed by spt75 mutations: an sptl5 his4-9175 /ys2-773R2 strain is His+ Lys-. To clone SPT75, strain FW1259 (MATa sptl5-21 his4-9176 lys2-173R2 ura3-52 trplA7) was transformed with a yeast library in the plasmid YCp50 (Rose et al., 1967) and approximately 9000 Ura+ transformants were screened for those that had acquired a His- Lys+ phenotype. Three different clones were isolated that contained common restriction fragments. To verify that the clones actually contained the SPT15 gene, a Clal-Xhol fragment from plasmid pFW213 (Figure 1) was subcloned into the integrating vector pRS306 (Sikorski and Hieter, 1969), producing the plasmid pFW216. The insert fragment was shown to direct plasmid integration to the SPT75 locus; the integrated plasmid, marked by URA3, was shown to segregate as an allele of spt15 for two independent transformants of strain FW1091 (SpT75) when crossed by strain FW1107 (spt75-735).

Three lines of evidence demonstrate that the SPT15 gene is the same as the gene encoding the TATA binding factor TFIID as isolated by Hahn et al. (1969a). First, physical analyses demonstrate that the SPT75 clone and the TFIID clone of Hahn et al. (1969a) contain the same piece of DNA. Construction of subclones from one of the initial SPT75 plasmid clones led to identification of a 2.4 kb EcoRI-BamHI restriction fragment that complemented an spt75 mutation (Figure 1). Restriction digests of the SPT75 plasmid pDE32-1, which contains this insert, and the TFIID encoding plasmid pSH216 (S. Hahn, unpublished data) yielded the expected comigrating restriction fragments. The two clones were identical for the presence or absence of 17 restriction sites, and the locations of all common sites were the same in both genes. In addition, Southern hybridization analysis, using both clones as probes, demonstrated that the two clones hybridize to one another and to a single S. cerevisiae genomic restriction fragment of the same size (Figure 2). We do not yet know if the faint bands observed in the hybridization to genomic DNA are due to hybridization to SPT75 sequences or to flanking sequences present in the probe. We detected no hybridization to a genomic digest of Schizosaccharomyces pombe DNA (Figure 2) or to genomic DNA from mouse, human, chicken, or Drosophila cells (data not shown). Second, we have shown that the TFIID-encoding clone of Hahn et al. (1969a) complements an spt75 mutation (Figure 3). Plasmid pSH226 (S. Hahn, unpublished data)

Yeast TFIID mutants 1185

pDE32-

Probe :

1

Figure 2. Southern SPTlC

pm218

Hybridization

Analysis

of

Southern hybridization analysis was done using the SPT75 probe pDE32-1 or the TFIIDencoding probe, pSH219 (5. Hahn, unpublished data). Plasmid or genomic DNA samples used are shown above each lane. Plasmids used (and the approximate amount of DNA loaded for each) are: pDE2C2, an SPT75 clone (35 ng); pSH225, a TFIID clone (14 ng) (S. Hahn, unpubtished data); and pDE34-5, which contains spf75-7OO::URA3 (3.5 ng). Genomic DNAs (and the approximate amount of DNA loaded) are: strain FY22, wild-type (1.5 ng); DE6, a diploid heterozygous at the SpT75 locus (SPT7%pf75-7OO::URA3) (0.2 pg); and FWP7l, an S. pombe strain (0.9 ng). All DNAs were digested with EcoRl and BarnHI.

was transformed into an spt75 mutant, strain FW1259, by selection for Ma+. All Ura+ transformants also became His- Lys+ (the phenotype conferred by the wild-type SPT75 gene). This experiment demonstrates that the clone isolated by Hahn et al. (1989a), based on yeast TFIID amino acid sequence, also encodes SPT75 function.

complete

Finally, we have shown genetically and physically that the open reading frames encoding TFIID and the SPT75 gene are the same. We constructed an sptl5 null allele in vitro, by deletion of the Xbal-Hindlll fragment within the SF775 gene (Figure 1). At the position of the deletion we inserted the yeast URA3 gene, creating the spf75 null al-

-histidine

pRS316

pDE28-6

pRS316

pDE28-6

pSH226

pDE34-6

pSH226

pDE34-6

Figure 3. Complementation

of spt75-27

by SF775 and TFIID Clones

The spf75-21 mutant Fw1259 was transformed by pRS316 (vector containing no yeast DNA insert), pDE29-5 (containing the SPT75 gene), pSH226 (containing the TFIID open reading frame identified by Hahn et al., 1999a), and pDE34-9 (containing the spt75 null allele, spt75-7OO::URA3). Transformants were grown in patches on synthetic complete media and then replica plated to a plate lacking histidine (- histidine) and then to a plate containing all amino ackl requirements (complete).

Cdl 1186

ura3

P

I

*

trp2 I

sp115 I

lad4

I

l&e, spt75-700::URA3 (plasmid pDE34-6). This null allele, when transformed into strain FW1259, fails to complement an sptl5 mutation (Figure 3). We determined the DNA sequence of SP775 in the region we deleted and found that the sequence was identical to base pairs 1715 to 1915 within the TFIID open reading frame determined by Hahn et al. (1969a). This result shows that the open reading frame that encodes TFIID is the same as the SpT75 gene. SP7Y5 Is Essential for Growth To determine the spn5 null phenotype, the spf75 null allele, spf75-7O&URA3 (described in the previous section), was integrated by homologous recombination into diploid strain BM61, resulting in a strain heterozygous for the disruption at the SpT75 locus (Figure 2). This diploid, DE6, was sporulated and tetrads were dissected; the germination pattern demonstrates that SPT75 is essential for growth. Tetrads segregated 2:2 for viability (10 out of 10 tetrads), and all viable spores were SP775+ (His- Lys+ Ura-). To confirm this result, we also transformed diploid DE6 with plasmid pDE40-4, which is an autonomous centromere-containing plasmid that contains the SPT75 and TRP7 genes. Tetrad analysis after sporulation of this transformant demonstrated that every Ura+ spore that formed a colony was also Trp+, and therefore contained the SPT75 plasmid. Furthermore, the TffP7 SPT75 plasmid was essential for growth in this genetic background, since Ura+ Trp- segregants were never detected during mitotic growth. Therefore, SPT75 is essential for growth. WI75 Is a Pmviously Unidentified Gene The SPT75 gene was genetically mapped in two steps. First, we localized the gene to chromosome V by Southern hybridization analysis of separated yeast chromosomes

Table 1. sptld

Suppression

Pattern SPT15

his491 7 him&9 176 hid-972 hid-9126 1~~2-128 1~~2-1286 lys2-173R2

spt15 + + + +

+

Suppression of Ty and solo 6 insertion mutations in sptl5 mutants. Symbols indicate growth on media lacking histidine for the his4 alleles and lacking lysine for the lys2 alleles. (+) growth; (-) no growth. Suppression was determined by tetrad segregation patterns in crosses behveen a parent that contained an sptl5 mutation and one that contained the relevant his4 or ly.92 insertion mutation.

Figure 4. Genetic Map Position of SfTl5 The S/775 gene was mapped to the right arm of chromosome V as described in the text. The gene maps 35 CM from TRP2 (7 parental ditypes, 0 nonparental ditypes, and 16 tetratypes) and 38 CM from RAD4 (12 parental ditypes, 1 nonparental ditype, and 15 tetratypes).

(see Experimental Procedures). Second, by tetrad analysis, spt75 mapped between trp2 and rad4 on the right arm of chromosome V (Figure 4). The genetic mapping of SPT75 demonstrates that it is a previously unidentified gene. Mutations in SP7Y5 Cause Pleiotropic Phenotypes Mutations in SPT75 were selected as suppressors of the insertion mutation his4-9776. Analysis of the suppression pattern of spt75 mutations with respect to a series of Ty and solo 6 insertion mutations at the HIS4 and LYS2 loci (Table 1) demonstrated that spfl5-27 confers a suppression pattern identical to that conferred by spt3, spti: and spt8 mutations. These three genes were identified in the same mutant isolation that identified spt75 (Winston et al., 1967). We also examined whether an SPT75 clone in a high copy number plasmid would cause suppression of insertion mutations, since this phenotype had been observed for certain other SPT genes (Clark-Adams and Winston, 1967; Clark-Adams et al., 1966; M. S. Swanson and F. Winston, unpublished data). In contrast to these other cases, a high copy number plasmid containing the wild-type SPT75 gene, pDE31-7, did not confer a suppression phenotype. In addition to suppression of insertion mutations, spt75 mutations confer at least three other mutant phenotypes. First, spt75 homozygous diploids fail to sporulate. This is true for at least two different spt75 mutations. Furthermore, an spt75 null mutation is partially dominant for a sporulation defect. For a diploid that contains SFW5 on one homolog and spt75-7OO:rURA3 on the other, sporulation was greatly decreased compared with the isogenic wild-type diploid. Restoring the SPT75 copy number to two in a heterozygous diploid by transformation with an SPT75 plasmid (pDE40-4) significantly increased sporulation levels (0.2% sporulation in strain DE6 transformed with vector alone versus 3.0% sporulation when transformed with pDE40-4). A second spt75 mutant phenotype is a mating defect when both parents are mutant. In a cross of spt75-27 by spn5-27 (strains FW1254 x FW1256), the number of diploids formed was greatly reduced compared with normal mating frequencies (data not shown). Crosses of spt75-27 strains by wild-type SfT75 strains showed normal mating frequencies. Finally, all sptl5 mutants grow slowly. These phenotypes taken together suggest that SPT75 is required for transcription of a large number of genes. Mutations in SP7’I5 Alter Transcription In Vivo Previous work has shown that Ty and solo S insertion mutations at HIS4 cause a His- phenotype because of alter-

Yeast TFIID mutants 1157

A

hls4.9126

HIS4 SPTlS+

b

l pt15-21

B

b

Hla-

Hh

htsl-9176

Ill”

I W

SPTlS+ -

spt15.21

HE4

his4-9176

Hlr-

Hl8+

his4-9126

tional for HIS4 expression because they contain translational starts and stops upstream of the HIS4 AUG. The his4-9126 and his4-9176 insertion mutations differ from one another in both the position and orientation of the 8 sequences with respect to the adjacent HIS4 gene (Farabaugh and Fink, 1980; Roeder et al., 1980). Both of these insertion mutations are suppressed from His- to His+ in an sptl5 mutant (Table 1). Northern hybridization analysis demonstrates that in an sp#5-21 mutant, transcription of hid-9726 and his4-9776 is altered (Figure 5C). For both insertion mutations, in SPTl5 strains (His- phenotype), we observed the previously identified longer transcripts. In both the sptl5-21 his4-9126 and sptl5-21 his4-9176 mutants (His+ phenotype), shorter transcripts are produced which migrate near the position of the wild-type HIS4 mFlNA. Therefore, mutations in SPT15 alter transcription at these loci by changing initiation from the 8 sequences to the normal HIS4 initiation site. Primer extension analysis shows that the shorter transcripts produced in the sptl5-21 mutants initiate at the same sites as wild-type HIS4 transcription (data not shown). Both Northern and primer extension analysis indicates that a small amount of Ginitiated transcription is still made in spt75-21 mutants, but that the majority of transcription initiation now occurs at the downstream site. The transcriptional changes observed in the sptl5 mutant are similar to those previously obsenred for an spt3 mutant (Figure 5; Silverman and Fink, 1984; Winston et al., 1984b). From these results we conclude that TFIID, encoded by SPTl5, is required for normal transcription initiation.

IRNA Discussion Figure 5. Transcriptional

Alterations

in sptf5 Mutants

Mutations in SPTf5 alter transcription of two different solo 6 insertion mutations in the 5’ noncoding region of HIS4: (A) his4-9726 and (6) his4-9775. The boxes with the arrows represent the S sequences; the direction of the arrows within the boxes indicate the normal direction of transcription from the S sequence. (In the case of his4-9176 the 6 sequence is in the opposite orientation from HIS4, yet transcription across HIS4 still occurs. The symbol TATA above the 9176 indicates a TATA consensus sequence on the strand transcribed in his4-977%) (C) For Northern hybridization analysis RNA was prepared from strains as described in Experimental Procedures and hybridized to the /f/S4 probe, pFW45 (upper panel). The amount of RNA in each lane was normalized by determining the amount of 1% ribosomal RNA present in each lane (lower panel). Approximately 15 trg of total RNA was loaded in each lane, except for the sample in lane 1, which contained 4 ug of RNA.

ations in transcription initiation. The insertion mutations, his4-9126 and his4-9176, cause a His- phenotype because of transcription initiation in the upstream 8 elements rather than at the normal HIS4 transcription initiation site (Figures 5A and 5B; Silverman and Fink, 1984; Winston et al., 1984b; Hirschman et al., 1988). For his49126, the longer transcript has been shown to initiate in the 6 at the normal initiation site for the Ty mRNA (Hirschman et al., 1988). For his4-9776, the size of the longer transcript is consistent with initiation in the 8 sequence as well. These longer transcripts are believed to be nonfunc-

Mutations in the SPT15 gene were previously isolated as suppressors of 8 insertion mutations in the promoter of the HIS4 gene. Here we have shown that the SPTl5 gene is the same as the gene that encodes yeast TFIID, as isolated by Hahn et al. (1989a). In addition, we have demonstrated that sptl5 mutations alter transcription in vivo. We directly examined the transcriptional effect caused by one sptl5 mutation; however, given the identical mutant phenotypes conferred by all three spf75 mutations, we think it is very likely that similar transcriptional alterations occur in all three spff5 mutants. Furthermore, we have shown that SPT75 is essential for growth and that sptl5 mutations are pleiotropic. From these results, we conclude that TFIID is an essential transcription factor, required for proper transcription initiation in vivo. Mutations in SPT75 were shown to suppress two different solo 8 insertion mutations at H/S4 by alterations in transcription. For both insertion mutations we observed the same type of transcriptional shift: initiation occurs in the upstream 5 in the wild-type SPT15 strains and shifts to a downstream initiation site in the sptl5 mutant strains. The simplest explanation for this shift in transcription initiation sites is that the wild-type TFIID protein preferentially acts at the upstream TATA; in the spfl5 mutant, the altered TFIID protein would now preferentially act at the downstream TATA.

Cdl 1188

Hirschman et al. (1988) demonstrated that this same shift in transcription initiation sites at his4-9126 could be caused by mutations in either the 6 or HIS4 TATA regions. This result suggests that there is normally a competition for one or more transcription initiation factors between these two promoter regions and that this competition is mediated by their TATA sequences. The data of Hirschman et al. (1988), taken with our current results on the transcriptional changes caused by TFIID mutations, suggest a model in which interactions between TFIID and different TATA regions can lead to different levels of transcription initiation. Results of Nakajima et al. (1988) have also suggested that TFIID can interact differently with distinct promoters. Hahn et al. (1989b) observed that, in vitro, yeast TFIID binds to several different consensus and nonconsensus TATA regions with high affinity. This result might appear to be contradictory to the model that mutant TFIID proteins alter transcription initiation by altering preference for different TATA regions. However, the altered specificity of a mutant TFIID protein may not be caused by a change in its binding to DNA. TFIID is only one component of a multicomponent initiation complex. The change in promoter specificity observed in sptl5 mutants may not result from an alteration in TFIID binding to DNA, but may result from altered TFIID interactions with other components of the initiation complex which may affect the interaction of the initiation complex with the TATA region. Altered TFIID proteins could also change interactions of the initiation complex with upstream promoter elements. Mutations in the histone genes HTAl and HTBl confer some of the same mutant phenotypes as sptl5 mutations, as loss of function mutations in these histone genes also suppress insertion mutations at the transcriptional level (Clark-Adams et al., 1988). Workman and Roeder (1987) have demonstrated that, in vitro, nucleosome assembly and TFIID binding compete with one another. In addition, studies of in vitro transcription of Xenopus 5S genes by the RNA polymerase III transcription complex has shown that transcription can be repressed by nucleosomes along with histone Hl (for a review, see Wolffe and Brown, 1988). These results, coupled with the results in this paper, suggest that the mutant phenotypes observed in histone and/or TFIID mutants may be caused by some change in the balance between TFIID binding and nucleosome formation around the two different transcription initiation sites. The sptl5 mutations were selected as suppressors of 6 insertion mutations; however, they are pleiotropic. They cause defects in sporulation and mating, and cause slow growth. These results indicate that the expression of a large set of genes is altered in these sptl5 mutants. Conceivably, all of the mutant phenotypes observed may result from altered specificity of TFIID: reduction of activity at some promoters and increased activity at others. However, it may also be true that there is an overall reduced TFIID activity in these mutants. Determination of the nature of the sptl5 mutations and the biochemical characterization of the mutant TFIID proteins that they encode should enable us to determine the nature of the TFIID

defects in these strains. In addition, isolation and analysis of spfl5 mutations by different genetic screens and selections will help to elucidate the cause of the mutant phenotypes. For example, it would be interesting to determine if a set of sptl5 mutations isolated in a different way would also cause suppression of insertion mutations. In summary, we have isolated mutations in SPTl5, the gene that encodes the TATA binding factor TFIID, and have shown that mutations in spf75 cause transcriptional changes in vivo. A large number of SPT genes have been identified, all by genetic selections similar to that used to isolate sptl5 mutations (Winston et al., 1984a, 1987; Fassler and Winston, 1988). Since there is a large body of evidence that TFIID is one of several components in the initiation complex for RNA polymerase II transcription (Fire et al., 1984; Hawley and Roeder, 1985, 1987; Burton et al., 1986; Reinberg and Roeder, 1987; Reinberg et al., 1987; Zheng et al., 1987; Buratowski et al., 1989), it seems possible that some SPT genes may encode other components of this complex. In addition, we expect that there are gene products that are essential for RNA polymerase II initiation that have not yet been identified by the in vitro transcription systems. The genes that encode such hypothetical factors may be identified by genetic analysis in yeast. The identification of genes that encode additional components of the initiation complex, along with further genetic and biochemical characterization of sptl5 mutants, should help to elucidate the role of TFIID in the process of transcription initiation. Experimental

Procedures

Strains The Saccharomyces cerevisiae strains used in this work are derived from S288C and are listed in Table 2. We used standard genetic nomenclature: uppercase letters denote a dominant allele; lowercase letters indicate a recessive allele. The allele SPTI5::pFw218 is an integration of plasmid pFW218 at the Sf775 locus. The mutation designated spI2-750 is a deletion that also removes at least part of the RAD4 gene. This mutation is scored by sensitivity to ultraviolet light. Escherichia coli strains HBlOl (Boyer and Roulland-Dussoix, 1989) and TBl (Bethesda Research Laboratories, Gaithersburg, MD) were used as hosts for plasmids. Media Rich (YPD), minimal (SD), and synthetic complete (SC) media were prepared as described by Sherman et al. (1978). For selection of Ura+ yeast transformants, SC medium lacking uracil was used. For scoring suppression of insertion mutations and other amino acid requirements, SD supplemented with the appropriate amino acids and SC media lacking the appropriate amino acids were used. Genetic Methods Mating, sporulation, and tetrad analysis were done as described by Mortimer and Hawthorne (1989) and Sherman et al. (1978). Yeast cells were transformed by the lithium acetate method (lto et al., 1983), and E. coli cells were transformed as described by Maniatis et al. (1982). The sptl5 null allele, sptk5-1OO::UffA3, was recombined into strains to replace the wild-type SfTT5 allele by transformation using a 3.8 kb EcoRI-BamHI restriction fragment isolated from plasmid pDE34-8 and selecting for Ura+ transformants. Plasmids Plasmids were constructed by standard procedures. In general, restriction fragments were purified from agarose gels by using low melting agarose (FMC, SeaPlaque) as described in Ausubel et al. (1988). Re-

;e

TFIID mutants

Table 2. Yeast Strains Strain

Genotype

FY22 FW219 RN825 FwQ89 FWlOQl Fw1107 FW1254 FW1258 FW1259 BM81

MATa MATa MATa MATa MATa MATa

DE5 DE8

024 L287 L443 L444

ura3-52 his3A200 ura3-52 ade2-101 spt3-101 hid-9126 lys2-173R2 urs352 hid-91 76 /y&l 73R2 urs3-52 spt15122 hi&91 7S IysZ-173R2 urs3-52 hid-9175 IysZ-173R2 ura3-52 trplA1 spt15135 MATa lys2-201 urs352 trplA1 sptlb21 MATa hid-9176 lys2-173R2 tQlA1 spt15-21 MATa hid-91 75 IysZ-173R2 ura3-52 tQlA 1 spt1521 MATalMATa hi&912SlhiH9125 lys2-1285llys2-1286 unr352luta3-52 bplA 1lhplA 1 leu2-3,112lleu2-3,112 MATa hid-9126 lys2-173R2 urs352 tQlA1 Sptlb21 MATdMATa hi.++-9126lhis4-9125 lys2-128Sllys2-1286 ora352lur&52 tQlA llttpl Al IeuZ-3,112lleu2-3,112 SPTl5lsptl51OO::URA3 MATa his3-532 MATa his4-9125 IysZ-173R2 IeuZ-1 spt3-202 MATa his4-9176 lys2-173R2 um3-52 spt3-202 MATa his4-9176 IysZ-173R2 ura3-52

with nick-translated DNA probes (Rigbyet al., 1977) in 4x SSC at 45°C. Washes were done as follows: two washes, each in 500 ml of 2x SSC at room temperature for 15 min; two washes, each in 500 ml of 2x SSC, 1% SDS at 45% for 20 min; one wash in 500 ml of 0.1x SSC at mom temperature for 20 min. Plasmids used as probes were the SP7Y5 plasmid pDE32-1 and pSH218 (S. Hahn, unpublished data). S. pombe DNA of strain FYP7l was provided by Charles Hoffman. The Southern analysis used to map SP775 to a yeast chromosome was done by hybridization in 4x SSC at 85%. The filter was washed four times in 0.1x SSC. 0.1% SDS, at 85%. The filter used was a gift from Michele Swanson and contained yeast chromosomes from strain YP148 (Vollrath et al., 1988) separated by electmphoresis in a contourclamped homogeneous electric field (CHEF) apparatus (Chu et al.. 1986).

Northern Hybridlzatlon Analysis Cells for RNA preparations were grown in SD medium supplemented with histidine. lysine, uracil, and tryptophan to a concentration of 10’ cells per ml at 3ooc. Totai RNA was prepared and subjected to electrophoresis as described (Carlson and Botstein, 1982; Winston et al., 1984b). Hybridization analysis was performed by using the dextran sulfate method described in the instructions for GeneScreen. Plasmids used as probes were pFW45, a HIS4 internal Bglll-Sall fragment in pBR322, and pSZ28, a ribosomal RNA probe (Szostak and Wu, 1979).

Acknowledgments striction enzymes and T4 DNA ligase were purchased from New England Biolabs and Boehringer Mannheim. Plasmid mini-preps were done by the boiling method as described by Holmes and Quigly (1981). Plasmid pFW213 contains a 10.1 kb SauM insert in the BamHl site of YCp50 (Johnston and Davis, 1984) and is the smallest of the three plasmids isolated from the library which complemented an sptl5-21 mutant. All subclones were generated from this plasmid. The plasmid pFW2f8 contains a 85 kb Clal-Xhol piece from pFW213 in the URA3 integrating vector p&308 (Sikorski and Hieter, 1989). The following plasmids were generated by subcloning the respective pieces from pFW213 into the polylinker of the CEN-ARS URA3 plasmid pRS318: (1) pDE18-1, 5.4 kb Clal-BamHI insert; (2) pDE19-1, 3.8 kb SamHI-Clal insert; (3) pDE23-8, 5.0 kb Hindlll insert; (4) pDE24-8, 4.4 kb Hindlll insert; (5) pDE28-8,2.4 kb EcoRI-BamHI insert. The plasmid pDE32-1 contains the 2.4 kb EcoRI-BamHI piece in the vector pUCl18 (Vieira and Messing, 1987). Plasmid pDE38-1 was used for sequencing and was made by digesting plasmid pDE32-1 with Hindlll and relegating, which resulted in a deletion of sequences in the SPfl5 gene from Hindlll to BamHI. Plasmid pDE40-4 contains the 2.4 kb EcoRIBamHl piece inserted into the polylinker of the CENARS TRW plasmid pSE358 (Elledge and Davis, 1988). pDE25-2 is a derivative of pFW213 that contains the sequences from the lefthand side of the insert to the first BamHl site, but with a deletion of an internal 0.5 kb EcoRl piece. Plasmid pDE34-8 contains the null allele of SPTl5, sptl5-lOO::URA3, and was constructed by deleting the 0.18 XbalHindlll fragment from pDE25-2 and inserting the URA3 gene subcloned from the pfasmid pMAlOQ3 (a gift from A. Hoyt and D. Botstein) as a 1.2 kb Xbal-Hindlll fragment. pDE31-7 is an insert of the 2.4 kb EcoRI-BamHI piece between the EcoRl and BamHl sites of the 2pm circle URA3 vector pCGS42 (provided by Collaborative Research). Plasmids pSH218 and pSH228 (S. Hahn, unpublished data) are the 2.4 kb RI fragment inserted into the vectors pSP85 (Prornega) and pRS318, respectively.

DNA Sequencing DNA sequencing was performed using double-stranded mini-prep DNA of the plasmid pDE38-1. Dideoxy sequencing was performed using techniques outlined in the Sequenase (TM) kit from U.S. Biochemical.

Southern Hybridization Analysis The Southern hybridization analysis (Southern, 1975) in Figure 2 was done by a modification of the procedure described by Roeder and Fink (1980). DNA was blotted to Genescreen (Dupont) in 4x SSC overnight, and the DNA was cross-linked to the Genescreen by ultraviolet light as described by Church and Gilbert (1984). Hybridizations were done

We are extremely grateful to Steve Hahn, Steve Buratowski, Philip Sharp, and Leonard Guarente for communication of unpublished data and for plasmids pSH218 and pSH228. We are also grateful to Karen Arndt and Betsy Malone for critical reading of the manuscript. We thank Jack Szostak for plasmid pSZ28. Richard Binari for Drosophila DNA, and David Deitcher for mouse, human, and chicken DNA. This work was supported by National Institutes of Health grant GM32Q87, National Science Foundation grant DCB8451849, and grants from the Stroh Brewing Company and the Lucille P Markey Charitable Trust, all to F W. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

July 20, 1989; revised July 25, 1989.

References Allison, L. A., Moyle, M., Shales, hf., and Ingles, C. J. (1985). Extensive homology among the largest subunits of eukaryotic and pmkaryotic RNA polymerases. Cell 42, 599-810. Ausubel, F M., Brent, R., Kingston, R. E.. Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1988). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and WileyInterscience). Bayer, H. W., and Roulland-Dussoix. D. (1989). A complementation analysis of the restriction and modification of DNA in E. coli. J. Mol. Biol. 41, 459-474. Buratowski, S., Hahn, S., Sharp, P A., and Guarente, L. (1988). Function of a yeast TATA element-binding protein in a mammalian transcription system. Nature 334, 37-42. Buratowski, S., Hahn, S., Guarente, L., and Sharp, P A. (lQ89). Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549-581. Burton, Z. F., Ortolan, L. G., and Greenblatt, J. (1988). Proteins that bind to RNA polymerase II are required for accurate initiation of transcription at the adenovirus 2 major late promoter. EMBO J. 5, 2923-2930. Cameron. J. R., Loh, E. Y., and Davis, R. W. (1979). Evidence for transposition of dispersed repetitive DNA families in yeast. Cell 18, 739-751. Carlson, M., and Botstein, D. (1982). Two differentially regulated mRNAs with different 5’ends encode secreted and intracellular forms of yeast invertase. Cell 28, 145-154.

Cell 1190

Cavallini, B., Huet, J., Plassat, J.-L., Sentenac, A., Egly, J.-M., and Chambon, P (1988). A yeast activity can substitute for the HeLa cell TATA box factor. Nature 334, 77-80. Chodosh, L. A., Oleson, J., Hahn, S., Baldwin, A. S., Guarente, L., and Sharp, P A. (1988). A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable. Cell 53, 25-35. Chu, G., Vollrath, D., and Davis, R. W. (1988). Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582-1585.

Lech, K., Anderson, K., and Brent, Ft. (1988). DNA-bound activate transcription in yeast. Cell 52, 179-184.

Fos proteins

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Matsui, T., Segall, J., Weil, P A., and Roeder, R. G. (lQ80). Multiple factors required for accurate initiation of transcription by purified RNA polymerase Il. J. Biol. Chem. 255, llQQ2-11996.

Proc.

Mortimer, R. K., and Hawthorne, D. C. (1969). Yeast genetics. In The Yeasts, Volume 1, A. H. Rose and J. S. Harrison, eds. (New York: Academic Press), pp. 385-480.

Clark-Adams, C. D., and Winston, F. (1987). The SPTG gene is essential for growth and is required for S-mediated transcription in Saccbaromyces cerevisiae. Mol. Cell. Biol. 7, 879-886.

Nakajima, N., Horikoshi, M., and Roeder, R. G. (1988). Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol. Cell. Biol. 8, 4028-4040.

Church, G. M., and Gilbert, W. (1984). Genomic Natl. Acad. Sci. USA 81, 1991-1995.

sequencing.

Clark-Adams, C. D.. Norris, D., Osley, M. A., Fassler, J. S., and Winston, F (1988). Changes in histone gene dosage alter transcription in yeast. Genes Dev. 2, 150-159. Corden, J. L., Cadena, D. L., Ahearn, J. M., and Dahmus, M. E. (1985). A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc. Natl. Acad. Sci. USA 82, 7934-7938. Davison, 8. L., Egly, J., Mulvihill, E. R., and Chambon, F! (1983). Formation of stable preinitiation complexes between eukaryotic class B transcription factors and promoter sequences. Nature 301, 888-888. Dignam, J. D., Martin, F? L., Shastry, 8. S., and Roeder, R. G. (1983). Eukaryotic gene transcription with purified components. Meth. Enzymol. 104, 582-598.

Reinberg, D.. and Roeder, R. G. (1987). Factors involved in specific transcription by mammalian RNA polymerase II: purification and functional analysis of initiation factors 118 and IIE. J. Biol. Chem. 262, 3310-3321. Reinberg, D., Horikoshi, M., and Roeder, R. G. (1987). Factors involved in specific transcription in mammalian RNA polymerase II: functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. J. Biol. Chem. 262, 3322-3339. Rigby, P W. J., Dieckmann, M., Rhodes, C., and Berg, t? (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 173, 237-251. Roeder, G. S., and Fink, G. R. (1980). DNA rearrangementsassociated with a transposable element in yeast. Cell 21, 239-249.

Elledge, E. S., and Davis, R. W. (1988). A family of versatile centromerit vectors designed for use in the sectoring-shuffle mutagenesis assay in Sacchammkces cetevisiae. Gene 70, 303-312

Roeder, G. S., Farabaugh, P J., Chaleff, D. T., and Fink, G. R. (1980). The origins of gene instability in yeast. Science 209, 1375-1380.

Farabaugh, P J.. and Fink, G. R. (lQ80). Insertion of the eukaryotic transposable element Tyl creates a 5-base pair duplication. Nature 286.352-356.

Rose, M. D.. Novick, P, Thomas, J. H., Botstein, D., and Fink, G. R. (1987). A Sacchammpss cefevisiae genomic plasmid bank based on a centromerecontaining shuttle vector. Gene 60, 237-243.

Fassler, J. S., and Winston, F. (1988). Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Sacchammpzes cerevisiae. Genetics 118, 203-212.

Samuel% M., Fire, A., and Sharp, F! A. (1982). Purification and characterization of a specific RNA polymerase II transcription factor. J. Biol. Chem. 267. 2003-2013.

Fire, A., Samuels, M., and Sharp, P A. (1984). Interactions between RNA polymerase II, factors, and template leading to accurate transcription. J. Biol. Chem. 259. 2509-2516.

Sawadogo, M., and Roeder, R. G. (1985). Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc. Natl. Acad. Sci. USA 82, 4394-4398.

Hahn, S., Buratowski, S., Sharp, P A., and Guarente, L. (198Qa). Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT75 suppressor of Ty element insertions. Cell, this issue.

Schena,

Hahn, S., Buratowski. S., Sharp, t? A., and Guarente, L. (1989b). Yeast TATA binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences. Proc. Natl. Acad. Sci. USA, 86, 5718-5722. Hawley, D. K.. and Roeder, R. G. (1985). Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II. J. Biol. Chem. 260, 8163-8172. Hawley, D. K., and Roeder. R. G. (lQ87). Functional steps in transcrip tion initiation and reinitiation from the major late promoter in a HeLa nuclear extract. J. Biol. Chem. 262, 3452-3481. Hirschman, J. E., Durbin, K. J., and Winston, F. (1988). Genetic evidence for promoter competition in Sacchammyces cerevisiae. Mol. Cell. Biol. 8, 4808-4815. Holmes, D. S., and Quigly. M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Anal. B&hem. 174, 193-197.

receptor

M., and Yamamoto, K. (1988). Mammalian glucocorticoid derivatives enhance transcription in yeast. Science 247,

985-987. Sherman, F., Fink, G. R., and Lawrence, C. W. (1978). Methods in Yeast Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 61-62. Sikorski, R. S., and Hieter, P (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in sacchamfnyces cemvisiae. Genetics 122. 19-27. Silverman, S. J., and Fink, G.R. (1984). Effects of Ty insertions on HIS4 transcription in Saccharomyces cemvisiae. Mol. Cell. Biol. 4, 12481251. Simchen, G., Winston, F., Styles, C. A., and Fink, G. R. (1984). Tymediated expression of the LyS2 and HIS4 genes of Sacchammyces cerevisiae is controlled by the same SPTgenes. Proc. Natl. Acad. Sci. USA 87, 2431-2434 Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Bill. 98, 503-517. Struhl, K. (1988). The JUN oncoprotein, a vertebrate transcription activates transcription in yeast. Nature 332, 844-850.

fac-

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J. Bacterial. 153, 183-188.

tor,

Johnston, M., and Davis, R. W. (1984). Sequences divergent GALI-GAL10 promoter in Sacchammps Cell. Biol. 4, 1440-1448.

Van Dyke, M. W., Roeder, R. G., and Sawadogo, M. (1988). Physical analysis of transcription preinitiation complex assembly on a class II gene promoter. Science 241, 1335-1338.

that regulate the cemvisiae. Mol.

Kakidani, H.. and Ptashne, M. (1988). GAL4 activates gene expression in mammalian cells. Cefl 52, 181-167.

Szostak, J. W., and Wu, R. (1979). Insertion of a genetic marker into the ribosomal DNA of yeast. Plasmid 2, 538-554

Vieira, J., and Messing, J. (1987) Production of single-stranded DNA. Meth. Enzymol. 753, 3-11.

plasmid

Yeast TFllD mutants 1191

Webster, N., Jin, J. Ft., Green, S., Hollis. M., and Chambon, l? (1988). The yeast UASo is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 tmns-activator. Cell 52, 169-178. Winston, F., Chaleff, D. T., Valent, B., and Fink, G. R. (1984a). Mutations affecting Ty-mediated expression of the MS4 gene of Sacchamnyxs cemvisiae. Genetics 107, 179-197 Winston, E, Durbin, K. J., and Fink, G. Ft. (1984b). The SP73 gene is required for normal transcripton of Ty elements in S. cerevisiae. Cell 39, 675-682. Winston, F., Dollard, C., Malone, E. A., Glare, J.. Kapakos. J. G., Farabaugh, F’., and Minehart, I? L. (1987). Three genes are required for tmns-activation of Ty transcription in yeast. Genetics 175, 649-656. Wolffe, A. F!. and Brown, D. D. (1988). Developmental 5s ribosomal RNA genes. Science 241, 16261632.

regulation of two

Workman, J. L., and Roeder, R. G. (1987). Binding of transcription factor TFIID to the major late promoter during in vitro nucleosome assembly potentiates subsequent initiation by RNA polymerase II. Cell 57, 613-622. Zheng, X.-M., Moncollin, V., Egly. J.-M., and Chambon, t? (1987). A general transcription factor forms a stable complex with RNA polymerase B (II). Cell 50, 381-388.