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Intragenic and Extragenic Suppressors of Mutations in The Copyright 0 1989 by the Genetics Society of America Intragenic and Extragenic Suppressorsof Mutations in the Heptapeptide Repeat Domain of Saccharomyces cerervisiae RNA Polymerase I1 Michael L. Nonet and Richard A.Young Whitehead Institute for Biomedical Research, Cambridge, Massachusetts02142, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02I39 Manuscript received April 1, 1989 Accepted for publication September 8, 1989 ABSTRACT The largest subunit of RNA polymerase I1 contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRBB partial deletion mutation (srb2A10) was constructed. The srb2Al0 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These pheno- types are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRBB gene encodes a factor that is involved in RNA synthesisand may interact with the CTR domain of the large subunit of RNA polymerase 11. HE largest subunits of eukaryotic, prokaryotic mouse (BARTOLOMEIet al. 1988), andD. melanogaster T and viral RNA polymerases share conserved (ZEHRINGet al. 1988). However, the function of the amino acid residues in eight colinear segments (re- CTR domain is not yet understood. In vitro transcrip- viewed by CORNELISSEN,EVERS and KOCK 1988; see tion experiments using purified RNA polymerase I1 Figure 1). The eukaryotic RNA polymerase I1 largest lacking the heptapeptide domain suggest that the do- subunit contains an additional domain at its carboxyl main is not required for proper transcriptioninitiation terminus consisting of multiple direct repeats of the in vitro (ZEHRINGet al. 1988; KIM and DAHMUS1989). consensus heptapeptide Pro-Thr-Ser-Pro-Ser-Tyr- Other experiments using antibody reagents directed Ser. The yeast largest subunit,encoded by RPBl, against the CTR domain suggest that it does function contains 26 or 27 repeats, depending upon the strain in initiation of transcription in vitro (DAHMUSand (ALLISONet al. 1985; NONET,SWEETSER and YOUNG KEDINGER 1983). Several clues may contribute to 1987), the Drosophilamelanogaster subunit contains deducing the function of the CTR domain. The hep- approximately 44 repeats (ALLISONet al. 1988; ZEHR- tapeptide domain has been shown to be phosphory- ING et al. 1988) and the mouse subunit contains 52 lated in mouse (CADENAand DAHMUS1987), and heptapeptide repeats (CORDENet al. 1985). In mouse evidence suggests it is also phosphorylated in yeast and yeast, 60% and 80% of the repeat units conform (SENTENAC1985). Finally, this domain is highly sus- exactly to the consensus sequence, while the remain- ceptible to proteolysis during purification (ALLISONet ing repeat units usually differ only at one position al. 1985; CORDENet al. 1985),and purified RNA fromthe consensus. The mouse repeat has been polymerase I1 frequently lacks the CTR. shown to function in place of the yeast repeat in vivo Several models for the functionof the heptapeptide (ALLISONet al. 1988). repeat domain in RNA polymerase I1 transcription The carboxy terminalrepeat (CTR) domain has have been proposed. These models postulate that the been demonstrated to be essential for RNA polymer- domain (1) interacts with trans-activating transcrip- ase I1 function in Saccharomycescerevisiae (NONET, tion factors, (2) serves to localize RNA polymerase in SWEETSERand YOUNG 1987; ALLISONet al. 1988), the nucleus, (3) acts as a “COW catcher” to transiently remove histones or other chromatin binding factors The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked“advertisement” from DNA during transcription, or (4) serves as a in accordance with 18 U.S.C. $1734 solely to indicate this fact. target for modifications that affect transcription in Genetics 123: 715-724 (December, 1989) 716 M. L. Nonet and R. A. Young A B CDE F G H E. coli 0' - RNAP I RNAP Ill 1 RNAP 11 t FIGURE2.-Parental plasmids. (Pro Thr Ser Pro Ser Tyr Ser ) (1975). Plates of all media types were supplemented with 2% agar (Difco Laboratories). 5-Fluoro-orotic acid (5-FOA) FIGUREI.-Structure of the largest subunit of RNA polymer- plates used to select against the presence of URA3 were ases. The structureof the largest subunit of RNA polymerase from made as described by BOEKE,LACROUTE and FINK(1 984). coli. ;md the largest subunit of RNA polynlerases I, 11, and 111 8. DNA manipulations: Yeast transformations were done from S. cerevisiae are compared in this diagram. The black boxes using a lithium acetate procedure (ITO et al. 1983). Cen- labeled A through H represent the eight regions of the largest subunit polypeptide where extensive amino acid sequence similari- tromere plasmids were isolated from yeast according to HOFFMANand WINSTON(1 987). DNA manipulations includ- ties have been found between the four RNA polymerase subunits. ing restriction digestions, ligations, CaC12Escherichia coli Similar results are obtained when the prokaryotic subunit is com- transformations, gel electrophoresis, and Southern analysis pared to the large subunit of other eukaryotic nuclear RNA polym- were performed essentiallyas described by MANIATIS, erases. The nomenclature system and a broad definition of the FRITSCHand SAMBROOK(1 982). Yeast DNA was isolated as regions is found in JOKERST (1987). The box with diagonal lines et al. represents the heptapeptide repeat domain found uniquely at the described by BOEKE (1 985). The YCp50 library of Sau3A partially digested genomic DNA of the s45 yeast carboxyl terminus of the RNA polymerase I1 subunit. n = 26 or 27 strain was created as described et al. (1987). It in yeast. depending on the strain, and 52 in the mouse. in ROSE contained approximately 20,000 individual recombinants general(AHEARN et al. 1987; ALLISONet al. 1985; with an average insert size ofapproximately 20 kb. For each of the RPBl mutations, a single strand of the DNAwas BARTOLOME! et d. 1988; CORDEN et a/. 1985; NONET sequenced using the double stranded plasmid sequencing et al. 1987). method of CHEN and SEEBURC(1 985) with some or all of a Temperature-sensitive and cold-sensitive mutants set of 27 20-nucleotide primers spaced at 200 nucleotide with defects in the heptapeptide repeat domain have intervals throughout the gene. been previously described in S. cerevisiae (NONET, RNA analysis: Total and poly(A+) RNA was isolated from yeast cellsaccording to ELDER,LOH and DAVIS(1 983). SWEETSERand YOUNG 1987b). The CTRdomains of Northern analysis was performed essentially asdescribed in RPBl in these conditional mutants contain 10 to 12 NONET et al. (1987). heptapeptide repeats, and the truncated domains ap- Plasmids: Plasmids are listed in Table 3. Parental plas- pear to affect the function of the enzyme rather than mids are shownin Figure 2. pRPl12 and pRPll4 were its stability. Here we describe the isolation and char- described in NONET, SWEETSERand YOUNG (1987). pRP1- 4, -5, -6, -10, -1 1, and -14 are mutant derivatives of the acterization of intragenic and extragenic suppressors pRPll4 (RPBl LEUP) centromere plasmid which carry the of conditional mutations in the repeat domain.Analy- rpbl-4, -5, -6, -10, -11,and -14 alleles, respectively (C.SCAFE sis of the intragenic mutations suggests interactions and R. A. YOUNG,unpublished data). The genomic DNA between the CTR domain and a separate segment of insert of pCTl is inserted in YCp50 such that the /3-lacta- RPBl, while investigation of the extragenic suppres- mase gene is transcribed in the opposite direction of the PUT2 gene. pCTl1, pCT 12, pCTl3, and pCTl4 were sor SRB2 suggests that theSRB2 product is a transcrip- created from pCTl by digesting with SacI, BamHI, BstEII, tion factor that may interact with the CTR domain. and SacII, respectively, and subsequently religating to delete portions of the genomic insert DNA. pCTl9 was created MATERIALS AND METHODS from pCTl1 in vivo by transforming strain N 1 14 with SacI digested pCTl1 DNA and isolating plasmid from a Ura+ Yeast strains and media: Yeast strains are listed in Table transformant which had gap-repaired the missingIO-kb 3. YPD mediumconsists of 2% yeast extract, 1% Bacto- SRBP region with wild-type SRBZ sequences from the chro- peptone (Difco Laboratories), and 2% glucose. YEPG plates mosome (ORR-WEAVER,SZOSTAK and ROTHSTEIN 1983). replace the glucose carbon source with 2.5% glycerol and pCT22 consistsof the 2.5-kb EcoRI fragment of pCTl 2..5% ethanol. Synthetic Complete medium (SC) consists of inserted into YCp50 such that SRBP transcription is counter 0.3% yeast nitrogen base without amino acids minus am- to transcription of the B-lactamase gene. pCT27 was made monium sulfate (Difco Laboratories), 1.O% ammonium sul- by inserting the 2.5-kb EcoRI fragment of pCTl9 into fate, 0.2% of an amino acid mixture described below, and pBLUESCRIBE I1 SK(+) (Stratagene) such that SRBZ tran- 2% glucose. Dropout medium minus selectable nutrients scription was opposite to LacZ' transcription. pCT30 was [DO - nutrient(s)] consists of SC medium lacking amino created by replacing the 550-bp Ncol fragment of pCT27 acids or nitrogenous bases. The amino acid mix consists of with a 1.1-kb Smal fragment containing the URA3 gene a mixture of 4 gleucine, 2 gof each ofthe 19 other standard (oriented in the opposite direction of SRBP).
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