Cell, Vol. 65, 349-357. April 19, 1991, Copyright 0 1991 by Cell Press

Dominant Negative Mutations in Yeast TFIID Define a Bipartite DNA-Binding Region Pranhitha Reddy’ and Steven Hahn Fred Hutchinson Cancer Research Center 1124 Columbia Street Seattle, Washington 98104

Summary Genetic analysis showed that the conserved Cterminal 160 amino acids of yeast TFIID contain all the essential functions for growth of yeast and response to acidic transcriptional activation signals. A genetic screen was used to identify functionally important residues within this C-terminal region. Five dominant TFIID mutations were isolated that had lost the ability to bind DNA. Four of these mutations were single amino acid substitutions in the most N-terminal of two 66-67 amino acid repeats in TFIID. Analogous mutations made in the most C-terminal repeat all failed to bind DNA and inhibited growth of cells, suggesting that the DNA-binding function of TFIID is partitioned between the two repeated regions. Overproduction of wild-type TFIID rescued the dominance of the TFIID mutants, suggesting that the mutant proteins are dominant because they compete with wild-type TFIID for binding to one or more essential transcription factors. Introduction Accurate initiation by RNA polymerase II requires at least five general transcription factors in addition to RNA polymerase (Matsui et al., 1980; Samuels et al., 1982; Sawadogo and Roeder, 1985; Reinberg et al., 1987; Zheng et al., 1987; Sopta et al., 1989). These factors, termed TFIIA, -6, -D, -E, and -F, can interact with polymerase and promoter DNA, forming a stable initiation complex (Van Dyke et al., 1988; Buratowski et al., 1989). Once this complex is formed, polymerase is capable of initiating mRNA synthesis. Insight into the process of transcription initiation and its regulation requires a detailed understanding of the interactions between the general transcription factors, polymerase, and promoter DNA. The TATA element-binding factor TFIID is essential for transcription initiation at many if not all polymerase II promoters. In vitro, purified TFIID binds to the TATA element and can nucleate the assembly of the polymerase II machinery into a stable initiation complex (Van Dyke et al., 1988; Buratowski et al., 1989). The function of TFIID has been highly conserved throughout evolution, as yeast TFIID will fully complement basal level transcription in a mammalian in vitrosystem (Buratowski et al., 1988; Cavallini et al., 1988). TFIID from Saccharomyces cerevisiae is a fIIOfIOfIwiC 27 kd polypeptide (Buratowski et al., 1988; Hahn et al., ‘Present address: Department of Pharmacology, ington, Seattle, Washington 99195.

University

of Wash-

1989a; Horikoshi et al., 1989a) and can promote transcription by binding to TATA elements with at least several different related sequences (Hahn et al., 1989a). TFIID binds DNA as a monomer (Horikoshi et al., 1990). The yeast TFIID gene was cloned (Hahn et al., 1989b; Eisenmann et al., 1989; Horikoshi et al., 1989b; Schmidt et al., 1989; Cavallini et al., 1989) and shown to be an essential gene (Eisenmann et al., 1989; Cavallini et al., 1989). The C-terminal 180 amino acids of TFIID contain two 66-67 amino acid repeats that share about 30% identity (Cavallini et al., 1989; Hoeijmakers, 1990; Nagai, 1990; Stucka and Feldmann, 1990). TFIID has been cloned from human, Drosophila, Arabidopsis, and Schizosaccharomyces pombe (Peterson et al., 1990; Fikeset al., 1990; Kao et al., 1990; Gasch et al., 1990; Hoffmann et al., 1990a, 1990b). The C-terminal 180 amino acids of all the TFllDs have been very highly conSeNed (80%-90%), while the N-terminal regions show little or no similarity. As demonstrated below, the conserved C-terminal 180 amino acids of yeast TFIID contain all the essential regions for both DNA-binding and transcription initiation function in yeast. Furthermore, the DNA-binding region is partitioned with residues in both repeats, contributing to DNA binding. Results The C-Terminal Domain Contains All Essential Function To test the function of the conserved C-terminal region of TFIID, several N-terminal deletions were constructed. These deletions were transcribed and translated in vitro and tested for DNA binding at the adenovirus major late promoter TATA element (TATAAA) using a native gel mobility shift assay. Deletion of up to 60 amino acids from the N-terminus of TFIID had no effect on DNA binding, while deletion of 71 amino acids abolished all detectable binding activity. In addition, a series of small (2-20 amino acids) internal deletions and insertions was constructed, which spanned the entire C-terminal 180 amino acids of TFIID. All of these were severely decreased in their ability to bind DNA in agreement with the results of Horikioshi et al. (1990). The fact that all small internal deletion and insertion mutations severely decreased DNA binding suggests that the C-terminal 180 amino acids comprise a structural unit in which precise positioning of amino acid side chains is necessary for folding and/or stability of the folded structure. To test whether the 180 amino acid C-terminal region contained all the essential functions of TFIID, the N-terminally deleted mutants were used to replace the wild-type TFIID gene in yeast by a plasmid shuffle strategy (Boeke et al., 1987). The deleted derivatives were placed in a low copy ARS CEN plasmid such that the TFIID gene expression was driven by the wild-type TFIID promoter. This plasmid, marked with LEUP, was transformed to a strain containing achromosomal TFIID deletion and a URA&marked plasmid containing the wild-type TFIID gene. The trans-

Cell 350

Al-71

--

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(I, :: t 0 3 (3

0) :: III

2 0

aJ z + 0 5

minimal and rich plates containing glucose galactose and lactic acid as carbon sources and were grown at 25%, 30°C, and 37% on synthetic glucose plates. No difference in growth rate between Al-60 and wild type was seen under any condition tested. The Al-60 cells were also competent to mate with a wild-type cell of opposite mating type. As expected, Al -71 was lethal and no survivors were seen on the 5-FOA plate. Western blot analysis with polyclonal TFIID antiserum directed against the N-terminal region confirmed the absence of full-length TFIID in the Al 60 strain (data not shown). Mutations in TFIID termed spt are known that can support the growth of cells but alter the start site of transcription at a subset of RNA polymerase II promoters (Eisenmann et al., 1989). Additionally, it was reported that deletion of the nonconserved N-terminal domain from human and Drosophila TFIID blocked the response to the activators Spl and CTF in an in vitro transcription assay (Peterson et al., 1990; Pugh and Tjian, 1990). To address these points for the mutant TFIID, mRNA was isolated from the Al-60 strain and transcription from three promoters was examined by primer extension (Figure 1 B). Like wild type, the Al-60 strain did not promote detectable mRNA synthesis from the GAL7 promoter in glucose medium. When strains were grown in galactose, both wild-type and mutant TFIID responded to the acidic GAL4 activator to promote identical levels of transcription from identical start sites. Similar results were seen at the CYC7 promoter, which responds to the acidic activators HAP1 and HAP2,3,4 (Pfeifer et al.. 1989; Forsburg and Guarente, 1989) and with the phosphoglycerate kinase promoter PGK(data not shown; Ogden et al., 1986) which responds to an uncharacterized activation signal

Isolation

GALI Figure 1. The Nonconserved Essential

CYCI N-Terminal

Region of Yeast TFIID Is Not

(A) Growth of yeast contaming TFIID deletions on synthetic complete glucose plates without leucine at 30%. Al-60 and Al-52 contain the amino acids MARG fused to amino acids 61-240 and 53-240 of yeast TFIID. (6) Primer extension analysts of mRNAs extracted from either wild-type (W.T.) or the Al-60 strarn.

formants containing two plasmids were streaked to plates containing 5fluoroorotic acid (5-FOA) to cure the URA’ wild-type TFIID plasmid. 5-FOA survivors were streaked to minimal plates without leucine and checked for the loss of the URA plasmid. As shown in Figure 1 A, strains containing TFIID deletions of amino acids l-52 and l-60 grew at the same rate as wild-type cells. Cells were tested on

of Dominant

TFIID Alleles

Since deletion analysis did not reveal any details of functional domains within TFIID, a genetic screen for TFIID mutants was next employed to identify important residues within the conserved region. Two types of mutations were sought. The first type would still bind DNA normally but would be defective in protein-protein interaction with another essential transcription factor. The second type of mutation would still be competent to carry out proteinprotein interactions but would fail to bind DNA. It was reasoned that both types of TFIID mutations would be dominant to wild-type TFIID when expressed at higher than wild-type levels. In the first case, mutant TFIID would bind the TATA elements at promoters and block access to the wild-type TFIID. In the second case, the mutant TFIID would sequester one or more essential transcription factors into nonproductive complexes. To screen for such mutants, the TFIID open reading frame was fused to the yeast GAL promoter to generate pSH229 containing a galactoseinducible TFIID gene. As measured by Western analysis, this plasmid overproduces TFIID about 20-fold compared with wild-type strains when cells are grown for at least five generations in galactose. Surprisingly, overproduction of TFIID at this level has no detectable growth phenotype nor does it affect either basal level or induced expression (unpublished data) from the

Dominant 351

Negative

TFIID

Mutations

only one out of about 200 lethal mutations gives a dominant phenotype. The fact that dominant TFIID mutations were rare led us to use a more efficient system for generation of additional dominant alleles. As detailed in Experimental Procedures, the UA!&-TFIID fusion was inserted in an ARS CEN yeast vector containing the phage fl origin of replication. Single-stranded DNA from this plasmid was used as a template for forced nucleotide misincorporation mutagenesis using reverse transcriptase (Laio and Wise, 1990). Using this method, mutagenesis frequencies in the TFIID coding sequence were typically 150%50%. New mutagenized libraries were screened as before, and four new dominant mutants were isolated that grew normally on glucose but slowly on galactose (Figure 4a). The mutant genes were sequenced, and all were found to be changes in the more N-terminal repeat region (Figure 3). These mutations are at residues that are conserved in all TFIID genes cloned to date. The mutation Val,,-+Glu has the strongest phenotype. After incubation for up to 10 days at 30°C only tiny microcolonies appeared on galactose medium. PheIIs+Tyr and a double mutant PheIIB and Phess, both changed to Leu, inhibited growth on galactose about as much as the original Thr-Lys mutation. As discussed below, both changes in the double mutant were required for the dominant phenotype. The weakest allele was ArgIos+Cys. In liquid galactose medium this mutant grew with a doubling time of 9.4 hr compared with 4.4 hr for wild type. The other mutants did not grow well enough in liquid galactose medium to measure reliable growth rates. On glucose medium, all the mutants grew at the same rate as cells overproducing wild-type TFIID. The amount of the mutant proteins expressed in vivo was determined by Western analysis. Stability of mutant proteins in vivo is often a good measure of correct protein folding (Parsell and Sauer, 1989). Transformants containing either wild-type or mutant TFIID under UASGAL control were grown in minimal raffinose medium and then induced with 2% galactose for 3 hr. Protein was extracted and TFIID detected using TFIID antiserum (Experimental Procedures). Wild-type TFIID was induced about 5fold over the 3 hr induction as compared with an uninduced control (all strains also have a wild-type chromosomal TFIID gene). Like wild type, all the TFIID mutants described above were similarly induced about 5fold, showing that they were as stable as wild-type TFIID. None of the mutant proteins could support detectable growth of yeast when substituted in placeof the wild-type TFIID gene by plasmid shuffle.

GAL. Figure

2. Isolation

of a Dominant

Mutation

in TFIID

Strain BWGl-7A(Guarente and Mason, 1963)containing the UASwLTFIID plasmid with either wild-type TFIID or the Thr,,,-Lys mutant. Cells were streaked on synthetic complete glucose or galactose plates without uracil and grown 3 days at 30%.

CYC7 UAS2 element (Guarente et al., 1984). However, we found that insertion of TFIID into a different UASG~~ expression vector that overproduced TFIID at least severalfold more than pSH229 severely inhibited cell growth on galactose (data not shown). Plasmid pSH229 was mutagenized to generate a library of 10,000 independently mutagenized plasmids. This library was transformed to a wild-type strain, and transformants were replica plated to both glucose and galactose plates. Of 20,000 colonies screened, one transformant was found that grew poorly on galactose but normally on glucose, as evident from the size of isolated colonies (Figure 2). Curing of the plasmid restored the ability to grow on galactose. The mutant TFIID gene was sequenced and found to contain a single C to A transversion changing ThrllP to Lys, which lies in the more N-terminal of the two 88-87 amino acid repeats (Figure 3). Although ThrIIP is not conserved between the two repeats, this mutation lies within the most highly conserved region of the repeats, and Thrlli! has been conserved in all TFIID genes cloned thus far (Peterson et al., 1990; Fikes et al., 1990; Kao et al., 1990; Gasch et al., 1990; Hoffmann et al., 1990a, 1990b). The single dominant allele isolated was a very rare mutation. From the efficiency of mutagenesis of the URA3 gene on the TFIID plasmid (a gene with a roughly similar size as TFIID) it is estimated that about 2% of all plasmids contained a lethal TFIID mutation. Of the 10,000 independently mutagenized plasmids screened, this suggests that

Figure 3. Dominant Mutations Lack DNA-Binding Activity

En f

67

LQNIVATVTLGCRX.0

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157

IQNI"GSCD"SFPIWGLAFSEGTFSSYEPELFPGLIY+VSP

.IllI,.

I

I.

.

5 =1a

.I

II

I..II

II

GAKSEDD 131

III

III

I.IllI

XI~IFVSGKIVLTGAKQP.ii

222

in TFIID

That

Shown is an alignment of the two 66-67 amino acid repeats in yeast TFIID. Upward arrows indicate mutations isolated with a genetic screen for dominants. The bracket indicates a double mutation with Pheas and Phella both changed to Leu. Downward arrows indicate site-directed mutations. As discussed in the text, all mutant proteins except ValIsI+Glu are stable in vivo.

Cdl 352

a

Figure 4. Domrnant N-Terminal Repeat

Mutations in the Fall to Bind DNA

More

(a)Additional TFllD dominant alleles isolated rn the ARS CEN UASGal-TFIID plasmid pSH277. Cells were streaked to synthetic complete glucose or galactose plates without leucine and grown at 30°C. (A) RI,,-C; (6) wild-type pSH277; (C) F,w-Y; (D)T,,z -K; (E)F,, FI,6-L double mutant; (F) V,,-E; (G) F,,6-L (not dominant). (b) Wild-type and mutant TFIID alleles as indicated were transcribed and translated in vitro and tested for binding to the adenovirus major late promoter TATA element by a gel mobility shift assay Control lysate was not programmed with mRNA.

G GLUCOSE

F GALACTOSE

E

b

TFIID Mutants Are Defective in DNA Binding To test whether the mutant TFllD proteins could still bind DNA, the mutants were cloned downstream of the phage SP6 polymerase promoter and transcribed and translated in vitro. Unlike wild-type TFIID, which bound the TATA element from the adenovirus major late promoter, none of the mutants bound DNA detectably (Figure 4b). To examine the DNA-binding defect more closely, the ThrItn+Lys mutant was expressed in Escherichia coli under control of the phage T7 promoter. This mutant was expressed to the same steady-state level as wild-type protein, again suggesting correct protein folding. The mutant was purified from E. coli using heparin, DEAE, MonoS, and Superose 12 chromatography and was found to chromatograph exactly as wild type. A lOO-fold increase of purified mutant protein over the level of wild-type TFIID necessary to observe a gel mobility shift at the major late promoter TATA element showed no detectable binding (J. Ranish and S. Hahn, unpublished data). Taken together, our results suggest that the dominant mutations have produced a localized change in the protein that either directly or indirectly disrupts the DNA-binding surface of the protein. Dominant and Wild-Type TFIID Compete for the Same Target One model for the dominant phenotype is that the mutant

and wild-typeTFllDs are competing for essential transcription factor(s). An alternative model is that the mutant protein has acquired an abnormal function unrelated to wildtype TFIID and is toxic to the cell. To rule out this latter model, wild-type TFIID on a high copy number 2u plasmid was introduced into the strains containing the dominant mutations under control of the GALuAs. In wild-type strains, the high copy number plasmid overproduces TFIID at least 5-fold (unpublished data). It was found that this level of overproduction of wild-type TFIID can completely rescue the dominant phenotype of all the mutants. This shows that the mutant proteins are not toxic and strongly suggests that the mutant and wild-type proteins are competing for the same essential transcription factor(s). Both Changes in the Double Mutant Are Required for the Dominant Phenotype As described above, one of the mutants changed both Phe, and PheI16 to Leu. Phej16 was already identified as a critical residue for DNA binding because mutation of this amino acid to Tyr was also found to generate a dominant mutant that failed to bind DNA (Figure 4b). To test whether both changes in the double mutant were required for the dominant phenotype, Phe, and Phells were individually mutagenized to Leu. Surprisingly, neither change alone gave adominant phenotype. PheI16+Leu grew at the same rate as wild type on both glucose and galactose plates

Dominant 353

Negative

TFIID

Mutations

a

Figure 5. Dominant C-Terminal Repeat

Mutations

in the

More

(a) TFIID dominant alleles generated by sitedirected mutagenesis in pSH277. Cells were streaked to synthetic complete glucose or galactose plates without leucine and grown at 30% (A) Vls,+E; (B) T1,2+K (N-terminal mutant for comparison); (C) Fal+Y; (D) wild-type pSH277; (E) R,,+C; (F) V,+K; (G) F,-L (not dominant). (b) DNA-binding assay of site-directed TFIID mutants. Wild-type and mutant TFIID alleles as indicated were transcribed and translated in vitro and tested for binding to the adenovirus major late promoter TATA element by gel mobility shift assay. Control lysate was not programmed with mRNA.

GLUCOSE

(Figure 4a) even though it failed to bind DNA in vitro (Figure 4b), was stably produced in cells as measured by Western analysis (data not shown), and could not support growth when substituted in place of the wild-type TFIID gene by plasmid shuffle. Phes8+Leu gave a protein that could support normal growth of yeast when substituted in place of the wild-type TFIID gene. It seems likely that the PheIIs+Leu mutation produces a protein that has a defect not only in DNA binding, but also in association with one or more essential transcription components and thus does not inhibit growth of cells. We speculate that in the double mutant the two leucines interact either directly or indirectly through other residues to hold the protein in a conformation such that it can bind the other transcription factor(s) and lead to the dominant phenotype. Bipartite DNA-Binding Motif Deletion analysis of the C-terminal TFIID region discussed above and by Horikoshi et al. (1990) has demonstrated that the repeated regions in TFIID were not redundant. A likely alternative therefore is that both repeated motifs participate in the same function. To test this idea, mutations in the C-terminal repeat were made that were analogous to those isolated in the N-terminal repeat (Figure 3). The mutant proteins were expressed under GALUS control, and all were found to inhibit the growth of wild-type

cells (Figure 5a). Val=+Lys showed as strong a phenotype as the analogous ThrrIn+Lys mutation. ArgIps+Cys (doubling time 13 hr in galactose medium) showed a slightly stronger phenotype than the analogous Argros*Cys (doubling time 9.4 hr in galactose). Phem7-Tyr had a weak phenotype with a doubling time of 8.2 hr in galactose compared with 4.4 hr for wild type. Finally, ValIn+Glu had a weak and sometimes variable phenotype on plates with single colonies only slightly smaller than strains transformed with wild-type pSH277, but grew in liquid galactose medium with adoubling time of 10 hr. As expected from the PherIs+Leu mutant, which was not dominant, Phem,+Leu did not inhibit cell growth. Western analysis was used to measure the stability of the mutant proteins in vivo. With one exception, all the mutant proteins were induced approximately 5-fold by galactose, the same amount as wild-type TFIID. Synthesis of ValIs,+Glu, however, was not noticeably induced above background levels, showing that it was unstable. This explains why it has only a weak phenotype compared with the strong analogous mutation Val,,*Glu, which is stable. The ability of the mutant proteins to bind DNAwas tested by translation in vitro and DNA-binding assays (Figure 5b). None of the mutations in the C-terminal repeat bound DNA at detectable levels. These results suggest that residues critical for formation of the DNA-binding surface are split

Cell 354

TATATA --

@Q TATATA Figure 6. Two Models

for DNA Binding

by TFIID

In both models analogous parts of the repeated with DNA. The amino acids between the repeats oval. The N-terminal domain is not shown. (Top) repeat. (Bottom) Binding to overlapping direct

regions are in contact are shown as a striped Binding to an inverted repeats.

between the two repeated regions with at least several analogous amino acids cooperating in the same function. The one exception we found is the pair Val,, and Vah6,. As noted above, VallsI-Glu was unstable in vivo and probably did not fold into a stable structure. Thus, the failure of this mutant to bind DNA may not result solely from a perturbation of the DNA-binding surface of the protein. Discussion Deletion analysis of yeast TFIID in vivo showed that all essential function of the protein was contained within the evolutionarily conserved C-terminal region. This is in contrast to resultsobtained in vitro with human and Drosophila TFIID, in which deletion of the nonconserved N-terminal region abolished the response to two transcriptional activators (Peterson et al., 1990; Pugh and Tjian, 1990). Further deletion analysis of the conserved C-terminal region of yeast TFIID was not successful at defining functional domains within TFIID. This is probably because the C-terminal 180 amino acids comprise a structural unit whose precise folding is necessary for function. While only a few residues are probably involved in DNA binding for example, most internal deletions and insertions probably disrupt the overall structure necessary for protein folding or stability of the folded structure. To avoid this complication, yeast genetics was used to isolate mutations specifically defective in DNA-binding function. These mutations are dominant to wild-type TFIID and presumably act by titration of one or more essential transcription factors. Since the mutant proteins do not interact with DNA in vitro, we propose that TFIID normally interacts with one or more essential transcription factors before binding to DNA. One candidate for these factor(s) would be the class of acidic transcriptional activators, as it has been demonstrated that TFIID and a VP16 fusion protein interact in vitro (Stringer et al., 1990). Another class of candidates would be molecules termed coactivators,

which have been proposed to act as a bridge between DNA-binding activators and the general transcription machinery (Peterson et al., 1990; Pugh and Tjian, 1990; Berger et al., 1990; Kelleher et al., 1990). Finally, the mutant TFllDs may assemble with the rest of the transcription machinery to form a nonfunctional complex. In vitro experiments have shown that purified TFIID alone can bind to its target DNA sequence (Buratowski et al., 1988, 1989). After this protein-DNA complex is formed, the remaining general factors associate in a defined order. In vivo, however, it may be the case that TFIID and some or all of the transcription components are assembled before TFIID binds DNA. Further genetic and biochemical experiments with these mutants should lead to a better understanding of the interactions between TFIID and other factors both before and after DNA binding. Model for DNA Binding Dominant mutations in TFIID that have lost the ability to bind DNA are located in two separate regions of the protein at identical positions within the two direct repeat sequences. This suggests that unlike other known DNAbinding proteins, the DNA-binding function of TFIID is split into two separate regions of the protein. Our results do not exclude the possibility that other regions within TFIID besides the repeatsare involved in DNA binding. Deletions and insertions within the spacer between the two repeats affect DNA binding (Horikoshi et al., 1990; this study) as well as deletions in the region C-terminal to the repeats that is claimed to have a very weak homology to E. coli sigma (Horikoshi et al., 1989b). As TFIID structure is apparently very sensitive to amino acid substitutions, it is not yet clear whether mutation of these other two regions affects proper folding of the protein, stability of the folded structure, or only disrupts the DNA-binding surface. Figure 6 shows two models for DNA binding by TFIID, the simplest models consistent with our results. In both models analogous regions of the repeats are involved in contacting the DNA. This mode of DNA binding is very similar to how a dimeric protein binds DNA, but the difference in TFIID is that both monomeric subunits have been fused into one gene. As postulated earlier, this may have arisen during evolution by duplication of a gene containing only one repeated motif that bound as a dimer (Cavallini et al., 1989; Hoeijmakers, 1990; Nagai, 1990; Stucka and Feldmann, 1990). The difference between the two models is whether TFIID recognizes its target as a direct or inverted repeat. Most TFIID-binding sites are not perfect direct or indirect repeats; the sequences TATAAA and TAllTA are about as strong in binding as TATATA (Hahn et al., 1989a). Strong binding to asymmetric TATAs can be explained by the model because the TFIID repeats do not have identical sequence and probably do not make identical contacts with the DNA. This is in contrast to a typical dimeric protein in which both monomeric subunits have identical sequence and make very similar DNA contacts Unlike bacterial repressorsand gene-specific regulatory factors, which often function in an orientation-independent fashion, TFIID probably has a role in defining the direction

Dominant 355

Negative

TFIID

Mutations

of transcription initiation. This suggests a requirement for polarity in the binding of TFIID. The models shown in Figure 6 both show polarity in binding and would explain why asymmetric sequences such as TATAAA function to promote transcription of a downstream gene, while the reverse sequence llTATA does not (Nagwa and Fink, 1965). Function of the Nonconserved N-Terminal Region The difference in N-terminal sequence between the various TFllDs that have been cloned has led to the proposal that this region is involved in species-specific protein-protein interactions. Two reports suggest that the N-terminal region of human and Drosophila TFIID is involved in interaction with a molecule that acts as a molecular bridge between the activators Spl or CTF and TFIID (Peterson et al., 1990; Pugh and Tjian, 1990). In principle, the nonconserved N-terminal regions could also mean that this region serves no essential function. The results presented here support this latter model, at least for S. cerevisiae. Also consistent with this result is the fact that TFIID from S. pombe with a completely different N-terminal domain can complement a disruption in the TFIID gene of S. cerevisiae (Fikes et al., 1990). A. Berk and coworkers (CL Zhou et al., submitted) performed experiments similar to ours in testing the viability of yeast with deletions in the nonconserved N-terminal region of TFIID. In their yeast strains, deletion of the N-terminal 60 amino acids gave rise to cells that grew much slower than wild type; normal growth required amino acids 49-60 of the N-terminal region. After exchange of strains between our laboratories, Zhou et al. showed this difference to be due to our respective yeast strains. Therefore, in some strains, part of the TFIID N-terminal region is required for normal cell growth. Experimental

Procedures

Strains TFIID deletions were shufffed into a strain derived from BWGl-7A (Guarente and Mason, 1983) containing a chromosomal deletion of the TFIID gene from positions 1132 to 1910 (Hahn et al., 1989b) replaced with the yeast HIS4 gene. This deletion removes all but the last 14 amino acids of the chromosomal TFIID coding sequence.

TFIID

Mutations

N-terminal and C-terminal deletions were constructed in pSH227 (Hahn et al., 1989b) using Exo Ill and Sl nuclease (Erase-a-Base, Promega) with deletion endpoints containing an 8 bp Xhol linker. Appropriate deletions were recombined to regenerate in-frame deletions. The N-terminal deletions were reconstructed with a synthetic adapter that restored the initiation Met codon. Al -60 and Al-52 contain amino acids MARG fused to amino acids 61-240 and 53-240 of yeast TFIID. Most internal deletions contained l-4 amino acids inserted at the site of deletion due to the joining of the Xhol linkers. All constructions were verified by DNA sequencing. Mutations in the C-terminal repeat were created in plasmid pSH277 by oligonucleotidedirected mutagenesis (Kunkel et al., 1987). To isolate the Thr,,2+Lys TFIID dominant mutant, the TFIID open reading frame from pSH227 (Hahn et al., 1989b) was inserted into the UASGIL expression vector SDS-ATG (Guarente. 1983) to create plasmid pSH229. This plasmid was mutagenized in vitro with UV light and transformed into a UV-treated E. coli uvrA strain, which was enhanced in error-prone repair, following a method provided by T. Davis, U. Washington. Mutagenesis of the URAB gene on the same plasmid

was about 2% as monitored by transformation to a pyrF E. coli strain. A library of mutagenized plasmid was used to transform strain BWGl7A, and transformants were screened by replica plating for slow growth on synthetic complete galactose plates as compared with growth on glucose plates at 30°C. To mutagenize TFIID with forced nucleotide misincorporation, The UA& TFIID fusion construct was cloned to the ARS CEN vector PRS316(Sikorski and Hieter, 1989). which contains thephagefl origin of replication and a selectable LEU2 gene to create plasmid pSH277. Single-stranded DNA was made from this plasmid in a duf, ung E. wli strain. A library of mutant plasmids was then constructed using a modification of the forced nucleotide misinwrporation procedure (Laio and Wise, 1990). The forcing nucleotide wncentration was 2 mkt. and the concentration of the limiting nucleotides was 200 nM. Three oligonucleotides centered on positions 1630,1827, and 2000 (Hahn et al., 1989b) in the C-terminal region of TFIID were used as primers. Nucleotide sequencing analysis of six to eight plasmids isolated from each of the mutagenesis reactions showed that the single nucleotide substitution rate was 15%-50% when the forcing nucleotides were dATP and dlTP. Frameshift and multiple mutations occurred at 0%-15%. When using dGTP as a forcing nucleotide the mutagenesis rate was 90% with 50% of the plasmids having multiple mutations. The dominant double mutant was isolated from this reaction and contained three silent coding sequence changes as well as the double missense mutations. A total of 14,000 transformants was generated with plasmids mutagenized by misincorporation mutagenesis and screened as above for slow growth on galactose.

Expresslon

of Mutant

Proteins

and Western

Analysis

Levels of TFIID expressed in the dominant mutants were measured using Western blot analysis. Strain BWGl-7A containing either pSH277 with wild-type TFIID or each of the dominant alleles under control of the GAL promoter was grown in synthetic raffinose medium to an A, of about 1 .O. Galactose was added to a final wncentration of 2% to induce the expression of TFIID protein from the plasmids. Cells were harvested after 3 hr in galactose. Cells were lysed with glass beads, and soluble protein extracts were isolated as described (Pfeifer et al., 1987). Ten micrograms of protein from each sample was electrophoresed on SDS-PAGE and blotted to lmmobilon membranes. Western blots were incubated with polyclonal rabbit serum directed against TFIID protein. The blots were washed and incubated with 10’ cpm per 10 ml of ‘“l-labeled protein A and visualized by autoradiography. The results were quantitated by densitometry.

DNA-Blndlng

Assay

In vitro translated proteins were assayed for binding as described (Hahn et al., 1989b) except that the binding reactions contained 30 mM KCI, the nativegel bufferwas mMTris(pH 8.3), 190 mM glycine, 1 mM EDTA, and 5 mM magnesium acetate, and the gel was 6% acrylamide, 2.5% glycerol with the above Mg buffer and 0. 5 mM dithiothreitol. Gels were run at 4OC. This native gel system differs from one previously reported (Buratowski et al., 1989; Hahn et al., 1989b) by inclusion of Mg in the native gel and in the gel running buffer. As described by Horikoshi et al. (1989) this addition allows yeast TFIID to form a complex with DNA that is stable in the native gel.

Primer

Extension

Transcription start sites were mapped using AMV reverse transcriptase as described by Hahn et al. (1985) using oligonucleotide primers complementary to mRNA from the GAL7, CYC7, and PGK genes. Strains were grown to an A, of about 1 .O in synthetic medium containing 2% of the indicated carbon source. Total RNA was extracted and 20 ug was used for each extension reaction. Products were separated on 8% DNA sequencing gels.

Acknowledgments We thank Trishia Davis for her method of random plasmid mutagenesis, Jeff Ranish for technical assistance, Paul Sigler, Ron Reeder, David Auble. and Jeff Ranish for their comments on the manuscript, and A. Berk for communication of unpublished results. This work was supported by a grant from the National Institutes of Health and an

Cell 356

American Cancer Society Junior Faculty Award to Fellow of the Life Sciences Research Foundation. The costs of publication of this article were the payment of page charges. This article must marked “advertisement” in accordance with 18 solely to indicate this fact. Received

August

7, 1990; revised

February

S. H. P. R. is a Glaxo defrayed in pari by therefore be hereby USC Section 1734

26, 1991

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