Vol. 10, No. 12

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1990, p. 6123-6131 0270-7306/90/126123-09$02.00/0 Copyright C 1990, American Society for Microbiology

A Suppressor of an RNA Polymerase II Mutation of Saccharomyces cerevisiae Encodes a Subunit Common to RNA Polymerases I, II, and III JACQUES ARCHAMBAULT, KEITH T. SCHAPPERT, AND JAMES D. FRIESEN* Department of Genetics, The Hospital for Sick Children, 555 University Avenue, and the Department of Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada Received 29 May 1990/Accepted 3 September 1990

RNA polymerase H (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RP021, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RP021 (rpo214), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RP026, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo2l4 temperaturesensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RP026 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo214. These results support the idea that the RP026 and RP021 gene products interact. the catalytic subunit of bovine type 2A protein phosphatase and has been postulated to modulate the activity of RNAPII by altering the level of phosphorylation of the largest subunit of the enzyme. Suppressors of a temperature-sensitive mutation within the largest subunit of RNAPII have been isolated. Ten independent, extragenic suppressors belonging to a single complementation group were shown to be alleles of the KEX2 gene, which encodes a protease required for the processing of alpha-factor and killer-toxin precursors (31). From this study, it is clear that KEX2 can influence RNAPII, although the precise mechanism remains obscure. As part of a continuing series of genetic studies, we have isolated linker-insertion mutations within the gene encoding the largest subunit of RNAPII (RP021; J. Archambault et al., unpublished data). One allele in particular, rpo214, causes a rapid growth arrest in cells shifted to the nonpermissive temperature (Archambault et al., unpublished data). Pleiotropic phenotypes associated with this mutation include a partial requirement for inositol as well as a slow-growth phenotype at the permissive temperature. The inability to grow on medium lacking inositol has been documented for several mutations in genes encoding subunits of RNAPII (RP021, RP022, and RP024) (3, 50) and can be accounted for by a reduction in the level of transcription of the INOI gene, which encodes the enzyme inositol-1-phosphate synthase. As an approach to the identification of genetic loci affecting transcription by RNAPII as well as to gain a further understanding of the defect associated with the rpo214 temperature-sensitive mutation, we have isolated plasmids from a yeast genomic library that can suppress the rpo214 temperature-sensitive defect. One suppressor plasmid was shown to carry the wt RP026 gene, which encodes a subunit common to RNAPI, -II, and -III.

The high degree of conservation in both structure and function of the transcriptional apparatus of eucaryotes is well established. For example, it has been shown that transcription factors of higher eucaryotes, such as the TATA sequence-binding protein (TFIID) or CCAAT-binding proteins have functional homologs in the yeast Saccharomyces cerevisiae (12-14). In addition, extensive similarity exists among the three forms of RNA polymerase (RNAP) from a variety of organisms, including that of the single procaryotic enzyme (1, 2, 6, 10, 19, 46). In S. cerevisiae, RNAPII is composed of 10 subunits. The two largest subunits (the products of the RP021 [24, 48] and RP022 [48] genes, also called RPBJ and RPB2) are homologous to the bacterial ' and a proteins, respectively (1, 46). In addition, RNAPII contains eight smaller polypeptides, some of which are also subunits of RNAPI and RNAPIII (reviewed in reference 43). The well-developed genetics of S. cerevisiae makes it an attractive organism for the study of multiprotein complexes such as RNAPII. The isolation of mutations within the various subunits of yeast RNAPII has already begun to help establish their individual roles in transcription (2, 3, 22, 24, 34, 35, 41, 50) and promises to be a fruitful approach as other subunit genes are being isolated (39). A counterpoint to mutant isolation is the search for suppressors (26), which has already proven useful in identifying new proteins involved in the processes of transcription. Suppressors of a HIS4 transcriptional defect caused by the absence of the GCN4, BASJ, and BAS2 transcription activators identified four loci (SIT] to 4) (3). Two of these, SIT] and SIT2, are allelic to the genes encoding the two largest subunits of RNAPII. SIT3, either in its mutant form or as wild type (wt) in high copy number, can suppress the HIS4 defect in the absence of the TATA sequence. SIT4 encodes a protein homologous to

*

Corresponding author. 6123

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TABLE 1. Strains used Strain" Relevant genotype JAY212b .......... MATa rpo214 JAY435 .......... MATa rpo214(pRPO26) JAY430 .......... MATa rpo214(pRPO26dl) JAY429 .......... MA Ta rpo214(pRPO26d2) JAY431 .......... MATa rpo214(pRPO26d3) JAY427 .......... MATa rpo214(pRPO26d4) JAY432 .......... MATa rpo214(pRPO26d5) JAY433 .......... MATa rpo214(pJAY83) JAY436 .......... MATa(pRPO26) JAY444 .......... MA Ta RP026:: LEU2(pRPO26) JAY446 .......... MA Ta RP026:: LEU2(pRPO26)(pRPO26cDNA) JAY467 .......... MA Ta rpo214(pRPO26cDNA) JAY472 .......... MATa rpo214 RP026:: pJAY97 JAY476 .......... MATa RP026:: pJAY97 JAY490 .............. MATa rpo214 RP026:: pJAY97(prpo26-LI111) JAY491 .......... MATa rpo214 RP026:: pJAY97(prpo26-LR111) JAY492 ......... MATa rpo214 RP026:: pJAY97(prpo26-LI118) JAY493 .......... MATa rpo214 RP026:: pJAY97(prpo26-LK118) JAY494 ......... MATa rpo214 RP026:: pJAY97(prpo26-LR118) JAY495 .......... MATa rpo214 RP026:: pJAY97(prpo26-LI125) JAY496 ......... MATa rpo214 RP026:: pJAY97(prpo26-LK125) JAY497 ......... MATa rpo2-4 RP026:: pJAY97(prpo26-LI138) JAY498 .......... MATa rpo214 RP026:: pJAY97(prpo26-PA117) JAY499 ......... MATa rpo214 RP026:: pJAY97(prpo26-PA131) JAY500 ......... MATa rpo214 RP026:: pJAY97(prpo26-EK144) JAY501 ......... MATa rpo214 RP026:: pJAY97(pRPO26HA) JAY502 .......... MATa rpo214 RP026:: pJAY97(pFL39)

JAY503 .........

JAY504 .........

MATTa RP026:: pJAY97(prpo26-LI111) MATa RP026:: pJAY97(prpo26-LR111) MATa RP026:: pJAY97(prpo26-L1118) MATa RP026:: pJAY97(prpo26-LK118)

JAY505 .......... JAY506 ......... JAY507 ............ MATa RP026:: pJAY97(prpo26-LR118) MATx RP026:: pJAY97(prpo26-LI125) JAY508 . JAY509 . MATTa RP026:: pJAY97(prpo26-LK125) MATt RP026:: pJAY97(prpo26-LI138) JAY510 . JAY511 . MATot RP026:: pJAY97(prpo26-PA117) JAY512 . MATa RP026:: pJAY97(prpo26-PA131) JAY513 . MATot RP026:: pJAY97(prpo26-EK144) JAY514. MA T RP026:: pJAY97(pRPO26HA) MAT RP026:: pJAY97(pFL39) JAY515 . a All JAY strains have been constructed in our laboratory. They are all derived from W303-1A (MATa canl-100 his3-11,15 leu2-3,112 trpl-I ura3-1 ade2-1) or W303-1B (MATat canl-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 ade2-1). Both strains were obtained from R. Rothstein. b Characteristics will be presented elsewhere (Archambault et al., unpublished data). JAY212 is a derivative of W303-1A in which the RP021 gene has been replaced by rpo214.

MATERIALS AND METHODS Strains and media. The S. cerevisiae strains used are listed in Table 1. Growth media were prepared as described by Sherman et al. (44). When needed, 5-fluoro-orotic acid (5-FOA) was added at a concentration of 1 mg/ml (7). All plasmids were propagated in Escherichia coli JF1754 (22). Yeast transformation was done as described by Ito et al. (25). Plasmids. Plasmid pJAY83 is a derivative of pINT2 in which a ClaI site located near CEN3 has been filled in by using the Klenow fragment of DNA polymerase I. Plasmid pRPO26 contains a 2.1-kb XbaI-ClaI RP026 fragment (the ClaI site is derived from the pINT2 polylinker adjacent to the BamHI site that was used to construct the library) cloned into the unique XbaI and ClaI sites of pJAY83. Plasmid pRPO26HA contains this same RP026 fragment cloned into pFL39 (TRPI CEN6 ARS; from F. Lacroute). Plasmid pJAY97 contains a portion of the RP026 gene (Sau3A fragment from nucleotides 1026 to 1451) cloned downstream

MOL. CELL. BIOL.

of the GAL] promoter (27, 47) in a yeast integrative plasmid carrying the URA3 gene. Plasmid pRPO26cDNA (2,um TRPJ) has been isolated from a cDNA library described by McKnight and McConaughy (32). This library was constructed by cloning yeast cDNAs between the ADCJ promoter and the CYCI terminator in a yeast episomal vector (2,um TRPI ). A yeast integrative plasmid, pJAY86, carrying a deletion allele of RP026 (ARP026) was constructed for the isolation of RP026 by gap repair and excision. Plasmid pJAY86 (URA3 ARP026) carries an internal deletion in RP026 that spans two Bsu36I sites at nucleotides 230 and 1858. The 5' and 3' boundaries of the ARP026 allele are at nucleotides 1 and 2131, respectively. Characteristics of the rpo2-4 allele used in suppressor isolation. The rpo214 allele was isolated from a collection of mutant RP021 alleles generated in vitro by linker insertion mutagenesis (Archambault et al., unpublished data). The site of the mutation was determined by DNA sequencing. The rpo214 mutation was found to be the result of an insertion of a 12-bp XhoI linker (5'-CTCGAGCTCGAG-3') in the HaeIII site at position 3174 in the RP021 sequence (1). This mutation results in the insertion of four amino acids, Leu Glu Leu Glu, between amino acids 954 and 955 in the RP021 gene product. Yeast cells carrying the rpo214 mutation are temperature sensitive for growth and undergo one round of cell division after a shift to the nonpermissive temperature (37°C). A slow-growth phenotype and a partial inositol requirement at the permissive temperature (30°C) are also associated with the rpo214 mutation. Construction of yeast genomic library. To construct the plasmid library used in the isolation of RP026, DNA from yeast strain JAY357 was partially digested with the restriction endonuclease Sau3A, size fractionated on a sucrose gradient, and cloned into the BamHI site of plasmid pINT2 (URA3 CEN3 ARSI) (38); 40,000 independent bacterial colonies were obtained. More than 80% of the clones carry yeast DNA inserts ranging in size from 5 to 15 kb. Yeast strain JAY357 is a fast-growing derivative of JAY214 (MA Ta ade2-1 cannl-100 his3-11,15 leu2-3,112 trpl-J ura3-1 PGALrpo214). The pGAL-rpo2J4 allele has been constructed by replacing the endogenous RP021 gene by the rpo214 allele under the control of the GAL1O promoter. The characteristics of yeast strains JAY357 and JAY214 will be presented in more detail elsewhere (Archambault et al., unpublished data). Nucleotide sequence analysis. Both strands of the 2.0-kb KpnI-SpeI RP026 fragment were sequenced by the chain termination method (42), using a combination of deletions and commercially available primers or primers synthesized on the basis of the RP026 sequence. Single-stranded or double-stranded DNA templates were used. DNA manipulation and mutagenesis. Site-directed mutagenesis of RP026 was performed as described by Kunkel et al. (28). The RP026 mutant alleles thus created were subcloned into plasmid pFL39. Hydroxylamine mutagenesis of plasmid pRPO26HA was performed in vitro essentially as described previously (11). All DNA manipulations were done essentially as described by Maniatis et al. (30). Isolation of RP026 by gap repair and excision. Plasmid pJAY86 was linearized at the unique Bsu36I site and introduced into yeast cells. Repair and integration of the plasmid at the RP026 locus were confirmed by DNA blot analysis for several transformants (data not shown). For two independent transformants, the plasmid integrated into the yeast chromosome was recovered into E. coli after digestion of the genomic DNA with SpeI as previously described (37).

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PHENOTYPES STRAIN JAY JAY JAY JAY JAY JAY JAY

RP026 FRAGMENT

435 430 429 431 427 432 433

Sequences remaining 1-2131 (pRP026) 1- 1889 (pRP026d 1) 1- 1023 (pRP026d2) 1-717 (pRP026d3) 1-273 (pRP026d4) 1448-2131 (pRP026d5) vector (pJAY83)

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FIG. 1. Suppression of the rpo214 temperature-sensitive phenotype by DNA subclones. Various deletions were created in the 2.0-kb suppressing fragment and were subcloned into plasmid pJAY83. These subclones were introduced into yeast strain JAY212 by selecting for Ura+ transformants. The transformants were tested for growth at 37°C. The bar indicates the region remaining in the subclone. The endpoints of the deletions were determined by DNA sequencing. +, Growth; -, absence of growth.

Nucleotide sequence accession number. The nucleotide reported here will appear in the EMBL, GenBank, and DDBJ nucleotide sequence data bases under accession number M33924. sequence

RESULTS Cloning of a suppressor of rpo214. A yeast genomic library was constructed (see Materials and Methods) and was used to transform a yeast strain carrying the rpo214 allele (JAY212) in order to isolate plasmids that can suppress the rpo214 temperature-sensitive mutation. Of 40,000 transformants obtained, 100 had acquired a temperature-resistant phenotype at 37°C. Plasmid DNA from 40 of these 100 clones was isolated in E. coli and retested for the ability to suppress the temperature-sensitive defect of strain JAY212 (rpo214). Two of the 40 plasmids continued to display suppression; presumably the remainder of the original 40 temperatureresistant clones were revertants of the temperature-sensitive phenotype of strain JAY212. Restriction digest analysis of these two plasmids with HindlIl showed a common yeast DNA fragment of 1.5 kb which, when used as a probe, hybridized to both plasmids under high-stringency conditions. A common 2.0-kb KpnI-SpeI fragment encompassing the 1.5-kb Hindlll fragment was shown to carry the suppressing activity. Deletions within the 2.0-kb DNA fragment were constructed to determine which portions of the DNA inserts are essential for suppression. Deletion of sequences between residues 1023 and 1889 abolished suppression (Fig. 1), indicating that the suppressor gene must span this region. Nucleotide sequence analysis. The nucleotide sequence of the entire 2.0-kb KpnI-SpeI suppressing fragment was determined (Fig. 2). The sequence revealed a 155-amino-acid open reading-frame (ORF) interrupted by a 76-nucleotidelong intervening sequence. The putative intron contains the three highly conserved sequence motifs present in all yeast introns (21): the 5' splice junction GTATGT, the canonical branch point TACTAAC sequence near the 3' splice junction, and the 3' splice junction AG. The position of this intervening sequence at the amino terminus of the predicted ORF is also typical of yeast introns. The locations of the intron boundaries were confirmed by sequencing three idependent RP026 cDNAs isolated from the expression library constructed by McKnight and McConaughy (32). These three cDNAs were isolated by hybridization using the 1.5-kb

HindIII RP026 fragment as a probe. The position of the intron is indicated in Fig. 2. The location of the ORF is consistent with the results of the deletion analysis presented above. The 5' and 3' ends of the three cDNAs are also indicated in Fig. 2. The sequence of the ORF was identical to the sequence of the recently isolated gene encoding the 17,908-Da subunit of yeast RNAPII, which was cloned on the basis of partial amino acid sequence determination of this protein (49). We will refer to this gene as RP026, for the sixth-largest subunit of RNAPII. Examination of the predicted amino acid sequence of the RP026 gene product revealed three distinct regions. A very acidic region is located within the first 40 amino acids at the amino terminus of the protein, and a basic region is located in the middle portion of the protein between amino acids 61 and 97. These two regions are predicted to be very hydrophilic (29). The carboxy-terminal region of the RP026 gene product contains a heptad repetition of leucine residues. RP026 is a single-copy gene located on chromosome XVI. The copy number of RP026 in the yeast genome was determined by DNA blot hybridization of total genomic DNA (Fig. 3A). When the 1.5-kb HindlIl fragment was used as a probe for RP026, no difference was observed in the pattern of bands obtained under high-stringency or relatively low stringency hybridization conditions. This result suggests that a single copy of RP026 is present in the yeast genome. The chromosomal location of RP026 was determined by probing a DNA blot of yeast chromosomes that had been separated by pulse-field gel electrophoresis (provided by R. Wevrick and H. Willard). The RP026 probe hybridized in the region of chromosomes XIII and XVI (Fig. 3B). These two chromosomes were distinguished by reprobing the same DNA blot with a chromosome XIII-specific probe from the ADH2 gene (33) or with both probes simultaneously. On the basis of these results, the RP026 gene was assigned to chromosome XVI. RP026 is an essential gene. The availability of the cloned RP026 gene allowed us to determine whether this gene is required during vegetative growth. A disrupted allele of RP026 was constructed in vitro by replacing the sequences that lie between nucleotides 1023 and 1448 with the LEU2 gene (Fig. 4). By using a one-step gene disruption method (40), the chromosomal allele of RP026 was replaced with the

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ARCHAMBAULT ET AL. TCTAGAAAAAGACATATCAGGACATATATGACACTTCCAATTTTTGGTTACTAGTGAGTC GTCATGAATAATCATGTGCATTTGTAAACCATTTTCCCCCACACATGGTTTGCTACAAAT AGGACATTTTAATTTAGGATGATCATTTTTTATATGCGATTGCAATTGTGACCATATCCG AAACTCTTTGCAACAACCAGCAAAAGTACATTGGTAAGGATTTTCTACCTCAGGATCGTG ATGTTTAGAAATGTGATTTCTTAGCCTATATGGCCTCTGAAAGCTTTTATTGCAGTGTGG ACAGGTTAGTTTATGTAAATGAACAGATAAAATATGTGCCCTTAATTGTGGATGCTTGTA GAATCGGAGGTTGCATCCTTCCTCTGGACAAATGAAAGATTTGGTATGCGTTACTTCGTG TCGCTTCAGTTGCTGGCGAGTCGTCACTCCTTTTCCACAATAAGAACATTGGAATGGTTT CGTATCAGAATGCGTATACAAGTGTCTCTCTAAGTGACTCTTTTTAACGAAGGATTTTGC ACACTATCACACTGAAATGCTCTTAAACCCTGATGTACGCTTAATTGGTGTTCAGTCAAA ATTGAAGGTCTTGTAAATGCCTATCACAGCATCATAGTCACAGAAATATGTCTTTGGCCT ATTGCTCGATGAGCTTCTAGTTGATGTTAAACTGTTCAATGATTCTGAACTCTCTGATCG TGATATGGGAATGGTCTCCTGTTTAAGTTCGGCCAGTGGCATTCCTTCATTATTTAGAAC CTCTCCTCCCATTTGACAGCGATTTCAACAGTTACTACTGTGCTTAATGCCCTTATTTGA TAGTTAGTTCTTCTTATAATAAATAATCATTGTATATTGATTATTCGTATAGTATGATAT TTAATTGAAAAAAAAATTTTTTTTTTTTTAAGAATATCATTCAAAGGCATCAATCACAAC 1,2* 3* CTTGAAGAAAGGCTAAAAAGACACATTTTGCAGGTAACAGTGTAAAGATTAAGGCTACAA M S D Y E E A

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GAACTTCGAAGATTTTGATGTAGAGCATTTTTCTGATGAGGAGACTTATGAGGAAAAACC 1200 Q F K D G E T T D A N G K T I V T G G N TCAATTCAAGGATGGTGAAACAACCGATGCCAACGGTAAGACCATCGTTACTGGTGGTAA 1260 G P E D F Q Q H E Q I R R K T L K E K A TGGCCCAGAAGATTTTCAACAGCATGAGCAAATAAGAAGAAAGACACTTAAGGAAAAGGC 1320 I P K D Q R A T T P Y M T K Y E R A R I CATCCCAAAAGACCAAAGAGCAACTACTCCATACATGACCAAATATGAAAGGGCAAGAAT 1380 L G T R A L Q I S M N A P V F V D L E G TTTAGGTACAAGAGCCCTACAAATTTCCATGAATGCGCCAGTTTTTGTAGATTTAGAAGG 1440 E T D P L R I A M K E L A E K K I P L V TGAAACTGATCCATTGCGTATTGCCATGAAGGAGTTGGCTGAAAAGAAAATTCCTTTGGT 1500 I R R Y L P D G S F E D W S V E E L I V TATTAGAAGATATTTACCAGATGGTTCCTTTGAGGACTGGAGTGTGGAGGAACTCATTGT 1560 D L GGATTTGTGATTACACCTGCATTTTCCTTTATGTGTATTTGCTTTGTTTGTATATTATTA 1620 1,2,3* TAACTATAAAGAACGATTTATTTTGTAATGCAAATAACTGGACGAGAGAACCACGCTATA 1680 ACCTATTTGGTAATAAAAATGTTTGTATGAATAAAAAAGCTGCTGACTGCTTTCTTTGAT 1740

ATGTGTATATAGAATATTTATCGGTGATTGATCTAAGAAAAGCTTACCTCTGAAAGTTCT 1800 CTCTTCGTCGCTTGCTGATTTGTAGTAAACAATTTGAAAAGTTTAGCAAATTCTTCCTCA 1860 GGATTTTCAAAACCGGCTTCCTTTAAAGATCAATGACCTCATTCAAGGGAACGTTTAAAT 1920 CGTGTCCTCTTCCAATGGCTTTTAAACTTTCTTCCAAATTCTTCACGCTCGGGGAAACTG 1980 CGATAAGTTCTTCCCCATGATAGATAAGAATATGGGAAAGCTTACACCTTCTTCTCCAAC 2040 TTCCGCGGTACTGGTGTCATTGTCAGGTACCATTTTGGACCATTCCTCATCTGTCAATGT 2100 2133 CTTCCCCAATGTTGCATATATCTTGGTAAGATC FIG. 2. Sequence of the 2.0-kb KpnI-SpeI suppressing fragment and the predicted amino acid sequence of the RP026 subunit. *, Locations of the 5' and 3' ends of three RP026 cDNAs (1,2,3).

RP026::LEU2 allele in a strain carrying a plasmid-borne copy (URA3 marked) of the RP026 gene. We then determined whether cells that lost the plasmid-borne copy of RP026 retained viability. First, a transformant in which integration of the RP026 ::LEU2 allele had occurred at the chromosomal locus was chosen on the basis of DNA blot analysis. Cells of this strain (JAY444) were grown in nonselective medium for more than 10 generations and then tested for the ability to grow on medium lacking leucine and including 5-FOA (1 mg/ml). Only Ura- cells can grow on 5-FOA. No cells were recovered that could grow on medium containing 5-FOA (Fig. 4). Thus, loss of the plasmid containing URA3 and RP026 is lethal, indicating that the RP026 gene is essential. In contrast, when strain JAY444 was transformed with a second plasmid carrying an RP026 cDNA (JAY436), cells capable of growth on 5-FOA-containing medium were recovered. This result demonstrates that the RP026 gene product performs an essential function and that it likely does not belong to a family of functionally interchangeable proteins. Increased dosage of RP026 suppresses the rpo214 temper-

ature-sensitive phenotype. As mentioned above, the sequence of the RP026 ORF carried on the suppressor plasmid is identical to that reported by Woychik et al. (49). To confirm that the RP026 gene carried on the suppressor plasmid is of wt sequence over the entire length of the gene, we sequenced two independent copies of RP026 that had been isolated from a wt yeast strain by the method of gap repair and excision (see Materials and Methods). The sequences of both copies were determined (from nucleotides 50 to 2133) and compared with the sequence of the RP026 gene originally isolated by suppression of the rpo214 temperature-sensitive phenotype. In all cases, no nucleotide differences were found in the ORF or in the upstream (976 nucleotides) and downstream (567 nucleotides) untranslated regions. These results indicate that increased gene dosage of wt RP026 is sufficient to bring about suppression of rpo214. Increased expression of an RP026 cDNA suppresses the rpo2-4 temperature-sensitive phenotype. The observation that increased dosage of RP026 is sufficient to suppress the temperature-sensitive defect imposed by the rpo214 mutation prompted us to investigate whether increasing expres-

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presumably as a result of the overproduction of the RP026 gene product. To this end, a wt RP026 cDNA expressed from the promoter of the alcohol dehydrogenase 1 gene (pADC1) and carried on a high-copy-number plasmid (2Rm

TRPJ) was tested for its ability to suppress the temperature sensitivity and inositol auxotrophy of rpo2l4 mutant cells. Strain JAY467 carrying the RP026 cDNA acquired temperature resistance as well as the ability to grow on medium lacking inositol (Fig. 5), indicating that increased transcrip-

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FIG. 4. Disruption of the RP026 gene and effect on cell viability. (A) Structure of the disrupted allele of RP026. The open box represent the RP026 ORF. (B) Growth phenotypes of cells spotted on agar medium containing or lacking 5-FOA after 5 days of incubation at 30'C.

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Conditional expression of RP026. A yeast strain that expresses RP026 from the strong inducible promoter, PGALI' was constructed to verify that increasing expression of the .chromosomal RP026 gene results in suppression of the rpo214 temperature-sensitive phenotype. The GAL] promoter is induced 1,000-fold when cells are grown in medium containing galactose and is repressed in the presence of glucose (27, 47). The strategy used is depicted in Fig. 6. Plasmid pJAY97, which contains a truncated RP026 ORF fused to PGALIJ was used to transform JAY212 cells (rpo2l4), creating strain JAY472. Integration was directed at the RP026 locus by linearization of the plasmid with the restriction endonuclease AflII. Integration resulted in a duplication of the RP026 gene; a truncated RP026 ORF was placed under the control of the RP026 promoter, and a complete RP026 ORF was controlled by PGALI- As expected, growth of strain JAY472 (rpo214 RP026::pJAY97) was galactose 2 dependent. This strain is no longer temperature sensitive I i S w l -> despite the presence of the rpo214 mutation. This result demonstrates that suppression can be obtained by overexof FIG. 5. Suppression rpo2r4 phenotyrpes by increased exprespression of the wt RP026 gene at its normal chromosomal ie RP026 cDNA used to location, presumably as a result of overexpression of the transform yeast strain JAY212. The RP026 cDNA has been cloned downstream of the alcohol dehydrogenase promoter (ADCI) and RP026 gene product. In addition, strain JAY476 (RPO21 RP026:: pJAY97) was upstream of the CYCI transcription terminiator in a 2,um plasmid. constructed. This strain is also galactose dependent. The (B) Phenotype of strain JAY212 untransforr ned or transformed with the RP026 cDNA. Cells were spotted and incubated for 5 days on construction of strains that conditionally express RP026 agar medium at 30 and 37°C or on medium lacking inositol at 30'C. provides a rapid and efficient assay for determining the Strain W303-1A (wt) was used as a control effects of mutations in RP026, since cells can be transformed with mutant RP026 alleles and their phenotypes can be assayed on glucose-containing medium (see below). tion of a wt RP026 ORF is sufficient for suppression. This Isolation of a temperature-sensitive mutation in RP026. As result strongly suggests that supprevssion of the rpo214 a first step toward understanding the role of the RP026 gene mutation occurs through overexpressic:n of the RP026 gene product in the function of RNAPI, -II, and -III, a temperaproduct. sion of an RP026 cDNA. (A) Structure of th

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SUPPRESSOR OF AN RNA POLYMERASE

VOL. 10, 1990

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FIG. 7. Locations and phenotypes of point mutations in RP026. (A) Summary of the locations of the various point mutations within the RP026 gene and their phenotypes in combination with RP021 or rpo214 at 23°C. The RP026 gene product is diagrammed schematically. The filled box at the amino terminus represents the acidic region, the hatched box represents the basic region, and the bars at the carboxy terminus represent the heptad repeat of leucine residues. +, Growth; -, absence of growth. (B) Growth phenotypes of strain JAY472 (rpo214 RP026:: pJAY97) transformed with various RP026 alleles. The cells were spotted on agar medium lacking uracil and containing either glucose or galactose as the sole carbon source. The temperatures at which the cells were incubated are indicated.

ture-sensitive allele of RP026 was isolated by using a plasmid-shuffling strategy (7, 11). A single-copy replicating plasmid, pRP026HA (RP026 TRPI CEN4 ARSI), was mutagenized with hydroxylamine in vitro (see Materials and Methods) and was introduced into yeast strain JAY444 [trpl-l RP026:: LEU2 (pRP026)]. Approximately 8,500 Trp+ transformants were grown at 23°C on medium containing 5-FOA to select for cells that had lost plasmid pRP026. The 5-FOA-resistant colonies were then tested for growth at 37°C. One temperature-sensitive lethal mutation in RP026 was isolated that carried a G-to-A transition at nucleotide 1532, resulting in a glutamic acid-to-lysine substitution at amino acid 144 in the RP026 gene product. This mutation is recessive, as shown by the ability of a wt RP026 gene to rescue the temperature-sensitive phenotype (data not shown). Synthetic lethality of RP021 and RP026 mutations. To determine the role, if any, of the heptad repetition of leucines in the carboxy end of the RP026 gene product, these residues were mutated in vitro to isoleucine, lysine, or arginine. As well, two proline residues located within the heptad repetition of leucines were changed to alanine residues. The positions of these mutations and their phenotypes are summarized in Fig. 7. None of the mutations had any discernible effect on RP026 function; all of the mutant alleles were able to complement the RP026 LEU2 null allele or the PGALI-RPO26 allele (data not shown). Complementation was detected at 23 and 37°C on medium both containing and lacking inositol. The mutations that had been constructed in the leucinerepeat region of the carboxy end of RP026 (see above) were transferred to a strain containing the GALI-RP026 and the rpo2l4 alleles and were tested for growth. On glucose-

containing medium, growth of this strain was dependent on productive interaction of the two mutant gene products. Some mutant rpo26 alleles (LK118, LR118, LK125, LH138, and EK144) were inviable in combination with rpo214 (Fig. 7). This phenomenon, the combination of two mutations which by themselves are not lethal resulting in cell death, has been termed synthetic lethality (18, 45); it is a genetic indication of interaction of the two gene products. It is interesting that the leucine mutations that were furthest away from the carboxy terminus (LIlll, LR111, and L1118) showed no synthetic lethality. As the site of the rpo26 mutation moved closer to the carboxy terminus, growth in the presence of rpo214 was increasingly impaired (LK118, LR118, LK125, and L1138), although at a reduced level when the mutation involved a conservative change (LI125). Taken together, these results point to a role for the carboxyterminal portion of RP026 gene product in interaction with RP021 gene product. Such an interaction might be direct or might occur through the contact of other proteins, such as other subunits of RNAPII. DISCUSSION

Eucaryotic RNAPs are complex enzymes made of two large subunits associated with several smaller polypeptides (reviewed in reference 43). We have begun a genetic analysis of S. cerevisiae RNAPII with the goal of defining the role of the individual subunits in transcription. To this end, we have constructed linker insertion mutations (Archambault et al., unpublished data) in the gene encoding the largest subunit of this enzyme (RP021). One allele, rpo214, displays a temperature-sensitive phenotype as well as a partial inositol requirement and a slow-growth phenotype at the permissive

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temperature. To gain a better understanding of the molecular defect underlying these various phenotypes, we have used a yeast genomic library to isolate plasmids that can suppress the rpo214 mutation. The suppressor plasmids might carry genes whose products interact functionally with the largest subunit of RNAPII. Here, we report the isolation and characterization of one suppressor plasmid which carries RP026, an essential, single-copy gene that encodes a 17,980-Da subunit common to all three RNAPs (49). A primary finding of this work is that increased expression of RP026 can suppress the phenotypes associated with rpo214. More specifically, we have shown that suppression of rpo214 can occur under the following conditions: (i) increased copy number of the RP026 gene by placing it on a low-copy-number, replicating plasmid (CEN ARS); (ii) increased expression of RP026 by placing an RP026 cDNA under the control of the ADCJ promoter on a high-copynumber plasmid (2,um); and (iii) increased transcription of the RP026 gene by placing it under the control of the strong GAL] promoter. In all three cases, we have determined that the RP026 ORF being expressed has wt sequence. These observations strongly support the suggestion that an increased amount of the wt RP026 gene product is sufficient to bring about suppression. They are consistent with two hypotheses: (i) the rpo214 mutation increases the dissociation constant (Kd) between the RP026 and RP021 gene products or between the RP026 gene product and another component of the RNAP complex, or (ii) the rpo214 allele encodes an unstable protein that can be stabilized as a result of forming part of a larger complex. In the latter case, overproduction of the RP026 gene product could shift the equilibrium toward complex formation and as a result stabilize the rpo214 gene product. The observation that increasing transcription of rpo214, by placing the gene under the control of the strong GALIO promoter, does not suppress the temperature-sensitive phenotype is relevant to both possibilities (data not shown) because it suggests that increased amount of the rpo214 gene product is not sufficient to bring about suppression, although we have not determined that increased transcription of rpo214 results in greater accumulation of the encoded gene product. Suppression by increased expression of a wt gene has been observed elsewhere. For example, Clark-Adams et al. (17) have screened for high-copy-number clones that can suppress solo a insertion mutations in yeast. With this selection, they have isolated the wt histone locus HTAIHTBI, which contains the genes previously identified genetically as SPTIJ and SPT12. In an effort to begin understanding which regions of the RP026 gene product are important for its function(s), a temperature-sensitive allele of RP026 was isolated by using random chemical mutagenesis. The temperature-sensitive mutation results in a glutamic acid-to-lysine substitution at codon 144. In addition, mutations were constructed in the carboxy end of RP026 in the region of a heptad repeat of leucines. Site-directed mutagenesis of these leucine residues indicated that they are not essential for vegetative growth. However, some of the mutations are lethal when combined with rpo214. Such synthetic lethality is a genetic indication of interaction between the RP021 and RP026 gene products that might involve the carboxy-terminal portion of the RP026 gene product. Other examples of inviable combinations of mutant and suppressor alleles have been observed for genes encoding components of the cytoskeleton in S. cerevisiae (36). The genetic analysis presented here has established that

MOL . CELL . B IOL .

the RP021 and RP026 gene products interact functionally; this is not surprising, since they are part of the same enzyme complex. Such an interaction might be required for the structural or functional integrity of RNAPII. It is of interest that both subunits have been implicated in DNA binding (9, 23); possibly a very basic region, located in the central portion of the RP026 protein, could interact with DNA. Another feature of the RP026 gene product that has emerged from the sequencing analysis is the presence of a highly acidic region at the amino terminus of the protein. This region has the potential to fold into an alpha helix (15, 20). The function of this domain is unknown. The presence of two highly charged regions within the RP026 gene product might provide an explanation for the size discrepancy between the predicted molecular weight of the protein (Mr 17,908) and the molecular weight observed on polyacrylamide gels (Mr 23,000). Alternatively, this difference might be due to phosphorylation of the RP026 gene product (4, 5, 8) or other posttranslational modifications. The availability of the cloned RP026 gene should prove useful in the analysis of transcription by RNAPI, -II, and -III. The isolation of a subunit of RNAPI, -II, and -III as a suppressor of a mutation in the largest subunit of RNAPII is an example of the use of classical and reverse genetics to characterize a functional interaction between two subunits of a eucaryotic RNAP. It reinforces the validity of this approach in the identification of gene products that participate in transcription. ACKNOWLEDGMENTS We thank our colleagues, especially Emmanuel Maicas and Alan Davidson, for discussions, Jason Zacks and Peter Warburton for technical assistance, and Jacqueline Segall for critical reading of the manuscript and constant support during the course of this work. We also thank Rachel Wevrick and Huntington Willard for providing a yeast chromosome blot. J.A. holds a studentship from the Medical Research Council of Canada. This work was supported by a grant from the Medical Research Council of Canada. LITERATURE CITED 1. Allison, L. A., M. Moyle, M. Shales, and C. J. Ingles. 1985. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell 42:599-610. 2. Allison, L. A., K.-C. Wong, V. D. Fitzpatrick, M. Moyle, and C. J. Ingles. 1988. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol. Cell. Biol. 8:321-329. 3. Arndt, K. T., C. A. Styles, and G. R. Fink. 1989. A suppressor of a H1S4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell 56:527-537. 4. Bell, G. I., P. Valenzuela, and W. Rutter. 1976. Phosphorylation of yeast RNA polymerases. Nature (London) 261:429-431. 5. Bell, G. I., P. Valenzuela, and W. J. Rutter. 1977. Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. J. Biol. Chem. 252:3082-3091. 6. Biggs, J., L. L. Searles, and A. L. Greenleaf. 1985. Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II. Cell

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