Copyright 0 1989 by the Genetics Society of America

Intragenic and Extragenic Suppressorsof 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 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 of CTR domain 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 (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic of CTR domain mutations. The SRB2 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 encoding components of the 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 initiationof 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 inyeast 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 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). amino acids, 2 g inositol, 2 g uracil, 0.5 g adenine, 0.2 g p- Isolation and characterization of mutants: To obtain aminobenzoic acid. Minimal mediumlacking inositol has suppressing mutations, independent single colonies of the been previously described in CULBERTSONand HENRY C1, C3, and C6 strains were grown to saturation in YPD, RNA Polymerase I1 CTR Suppressors 717 TABLE 1 - Ino Heptapeptide repeatdeletion mutants I1 Strain Allele Repeats Phenotypes

N16 rpblAl32 4317 Lethal" +In0 N56 rpblAl40 6317 Lethal" Nl5 rpblAl3I 9317 Lethal" c3 rpblAlO3 10.517 cs, ts, Ino- c1 rpblAlOl 1 I2I7 cs, ts, Ino- C6 rpblAlO4 1 1317 cs, ts, Ino- v5 rpblAll0 13317 wt FIGURE3.--lnositol auxotrophy ofRPEI CTR deletion mutants. v7 rpblAl I I 1 74/7 wt Phenotypes of mutants on medium containing or lacking 100 pM VS rpblAlO8 1 gfiI7 Wt inositol at 30" were assayed by testing similar numbers of cells in v2 rpblAlO7 2 1417 Wt each spot. v4 rpblAlO9 22517 wt V8 rpblAll2 2 5'17 Wt given RPBl allele numbers as listed in Table 3. Although 227 RPBI 27 Wt the 10 CTR duplication suppressors are formally double mutations, these RPBl mutations havebeen given single The strain nomenclature is that in NONET.SWEETSER and YOUNG (1987). RPBl CTR deletion endpoints that produce viable, condi- allele numbers which encompass both the deletion and tionally viable, and nonviable cells are designated V, C, and N, duplication in the gene to simplify the nomenclature. respectively. Carboxy-terminal deletion alleles are designated The extragenic suppressor mutations in the isolates s3 rpblAl00-rpblAl62. and s45 were given the allele designations srb3-I and SRB2- These cells are inviable on 5-FOA-containing medium; that is, I, respectively. The s3 and s45 strains were crossed to strains they require the presence of the plasmid containing the wild-type N398, N399, and N400 containing the rpblAlOl, rpblAlO3 RPBI gene. and rpblAlO4 mutations on URA3 CEN plasmids and the resultant heterozygous diploids screened for loss of the and 5 X lo5, 5 X lo6, or5 X lo' cells of each culture were LEU2CEN rpbl plasmid.Diploids derived from matings plated on YPD and incubated at 12" or 15" for 14 to 30 with the s3 strain remained cs, showing that srb3-1 is reces- days. Only a single aliquot was plated from any individual sive. In contrast, all heterozygous diploids derived from culture. Suppressors of the cold-sensitive phenotype arose matings with s45 grew at 12" (although less well than the at a frequency of approximately as assayed by colony s45 isolate), indicating that the SRB2-I mutation exhibits a formation at 12". Fifty-two individual suppressor isolates of semidominant suppressing phenotype. Tetrad analysisof various sizes were picked, colony purified, and re-tested. some of these diploids (s3 x N398, s45 x N399) demon- The isolates were named sl through s52, using odd numbers strated that each SRB mutant in each diploid segregated 2:2 for large colony isolatesand even numbers for small colony (>12 tetrads dissected/cross). Neither SRB mutation was isolates. In no case was more than one small and one large linked to the URA3 or LEU2 centromere plasmids present isolate picked from a single plate (and in this case they were in each cross. SRB2-I and srb3-I are unlikely to be alleles of given consecutive isolate numbers). sl through s18 were the same gene since the presence of the plasmid pCTl9 isolated from C1 (at 14 days) and s3 1 throughs40 from C1 (containing wild-type SRB2) in strain s3 does not abrogate (at 30 days), s19 through s30 from C3 (at 30 days), s41 the suppression phenotype of srb3-I. In order to test the through s52 from C6 (at 30 days). Six isolates did not re- allele specificity of SRBZ-I, the strains 226 and N422 were test as suppressors. Thirty-one additional suppressors were transformed with rpbl LEU2 centromere plasmids, patched probably petites as evidenced by their slow growth rate, on medium containing 5-FOA, and single Ura+ colonies inability to grow on glycerol/ethanol as a carbon source were tested for conditional phenotypes on YPD media. (YEPG plates), and white color. These were not further The pCTl clone of SRB2-I complemented the put2 mu- characterized. tation in strains MB668-6D and MB331-17A (BRANDRISS RPBl mutant plasmid was isolated from 15 suppressors 1983). The put2::HIS3 mutation was crossed intoa todetermine if the suppressor mutation was intragenic. rpblAlO3 background and subsequently crossed to aSRB2- Strain 226 was transformed with each plasmid individually, I rpblAlO3 strain todemonstrate that SRBZ-1 is tightly primary transformants were patched onto 5-FOA-contain- linked to put2 (16 PD:O TT:O NPD). ing plates to select against cells containing the wild-type URA3 RPBlcentromere plasmid (pRP112), colony purified, and finally tested for ability to grow at 12". In 13 of 15 RESULTS cases the suppressor mutation was demonstrated to be intra- genic. Ten of the 13 intragenic mutations were demon- CTR conditional mutants areinositol auxotrophs: strated tobe duplications residing in the heptapeptide repeat We previously reportedthe isolation of a series of coding region by restriction mapping. The repeat coding mutations which progressively delete larger portions region ofsix of these ten duplication mutations was se- quenced as described under DNA manipulations (see Figure of the 27 heptapeptide repeats at the carboxyl termi- 5). The RPBI coding region from each of the suppressor- nus of RPBZ (NONETet al. 1987). Cells carrying RPBZ containing plasmids was subcloned into a URA3 centromere deletion alleles which retain greater than 13 full re- vector to replace the coding region of a wild-type RPBl peat units are wild type in phenotype,those which gene and the new plasmids were demonstrated to retain the retain 10 to 12 are cold-sensitive (cs) and/or temper- suppressor mutations. The whole coding region of the three non-duplication intragenic suppressor mutations was se- ature-sensitive (ts), and those with less than 10 repeat quenced as described under DNA manipulations (see Figure units are inviable (Table 1). Further investigation of 6). The thirteen RPBl intragenic suppressor mutations were the CTR domain mutants reveals that, in addition to 718 M. L. Nonet and R. A. Young

TABLE 2 r&Lbud- CI 1-2-3-4-5-6-7-8-9-10-11-12 Classification of suppressors of heptapeptide repeat mutants U U s31 1-2-3-4-5-6-7-8/6-7-6-9-lO~ll-l2 +2 Mutant SS 1-2-3-4-5-6-7-6-9-10/7-8-9-10-11-12 +3 s9 1-2-3-4-5-6.7-8/6-7-S/6.7-8-9-lO-ll~l2 +4 Suppressor class CI C3 C6 C3 I-2-3-4-5-6-7-8-9-10-11 Petites IS I1 7 U U sZl,r27 1-2-3-4-5-6-7-8/6-7-8-9"11 +2 Extragenic 1 0 1 Intragenic duplication 2 2 6 2 C6 I-2-3-4-5-6-7-6-9-IO-11-12 1 0 2 U U Intragenic point mutant ss~ ~.2-3-4-5-6.7.~-9~10.l~.9-lO-ll.lO-11-12+5 The frequency of all suppressors together was 10"'. FIGURE5."DNA repeat structure of intragenic suppressors which partially duplicate the repeat domain. A simple representa- A. mutants B. suppressors c. plasmid linkage tion of the DNA encodingthe heptapeptide repeat domain of several mutants, and suppressors which elongate the repeat domain. 12O L is shown. The DNA encoding the heptapeptide repeat domain consists of 21-bp repeat units which differ from one another by 24O zero to five nucleotides (mostly translationally silent changes). hch wt C1 C3 C6 s3 s17 s43 s45 s43 s45 s49 s51 repeat unit is represented by a number identifying the repeat unit's position in the wild-type RPBl gene. The structuredrawn for each FIGURE4.-Phenotypes of representative suppressors of CTR suppressor represents the simplest structure (fewest noveljunctions domain mutants. The growth phenotypes of representative mutants created) which fits the sequence data. The number of repeat units at 12" and 24" were assayed by spot testing on YPD medium. The added to the heptapeptide repeat domain of each suppressor is name of the strains are listed below the photographs. A, Wild-type listed at the right. strain Z27 and starting mutants. B, A representative set of colony- purified suppressor isolates. C. Phenotypes of cells (rpblAl87 back- ground) containing RPEl mutant plasmids isolated from the desig- (226) to test if the suppression phenotype was linked nated suppressor strains. to the original RPBI deletion mutation. In 13 of 15 suppressors, the suppressor phenotype was linked to being cold- and heat-sensitive, the conditional mutants the plasmid, suggesting that the suppressor mutation are also auxotrophs (Figure 3, Table 1). Strains car- was intragenic (Figure 4C). The remaining two sup- rying the rpblAlOl, rpblAlO3, and rpblAl04 alleles pressors were identified as extragenic suppressors(see (Cl,C3, and C6, respectively) grow normally on below). standard synthetic minimal medium (which contains One class of intragenic suppressors are heptapep- small amounts of inositol) but are unable to grow on tide repeat duplications: All cells containing intra- medium which lacks inositol. In contrast, CTR dele- genic suppressors were ableto grow on medium lack- tion mutants which are fully viable (>13 repeats) are ing inositol at 30" and in rich medium at 12", al- capable of growing in the absence of inositol. The C1, though not all cells with suppressing mutations grew C3, andC6 mutantsare not auxotrophic forany other at wild-type rates. Restriction analysis of the cloned standard amino acid or nucleotide precursor as they intragenicsuppressor mutations identified an elon- grow onstandard yeast minimal medium supple- gated CTR coding region in 10 of 13 suppressors. mented with uracil. Sequencing of the CTR of six of these intragenic Isolation of suppressors of CTR deletion mutants: mutations confirmed that spontaneous partial dupli- Spontaneous suppressors of three of the conditional cations of various sizes account for the suppression CTR deletionmutations rpblAl01,rpblAl03, and phenotype(Figure 5). The partialduplications in- rpblAl04 (strains C1, C3, and C6) were selected on crease the repeat coding regionby 2 to 5 repeat units. rich medium at 12 " and 15". Of 52 original suppres- The 21-bp repeat units of the wild-type RPBl gene sor isolates (sl-s52), 46 were characterized and di- differ from one anotherslightly in sequence (by zero vided into three groups (Table 2,see MATERIALS AND to five nucleotides per repeat),allowing one to identify METHODS for details). Thirty-one of the suppressors which of the repeats are duplicated. Repeat units six are probably petites, as evidenced by their slow growth througheight were frequentlyduplicated. Surpris- rate, inability to grow on glycerol/ethanol as a carbon ingly, two of the suppressors, s9 and s5 1, underwent source (YEPG plates), and white color. These mutants multiple duplication events. suppress the cs phenotype of rpblAIO1, rpblAl03,or An intragenic point mutationalso suppresses the rpblA104 upon incubation for 20 to 30 days at 12". conditional CTR mutants: One intragenic suppressor They were not further characterized due to theirslow of rpblAlOl (s17) and two intragenic suppressors of growth rate. The suppression phenotype of the re- rpblAlO4 (s43 and s49) did nothave alterations in the maining 15 suppressors ranges from weak to strong heptapeptide repeat coding regionof the RPBl gene. (Figure 4, A and B). The plasmid encoding the RPBI The three non-duplication intragenic mutations were mutation was isolated from each suppressor strainand mapped to the coding region of the RPBl gene, and reintroducedinto a "wild-type" strainbackground sequenced. All three independently isolated mutations RNA Polymerase 11 CTR Suppressors 719 SrrsnNrr sEnnUGE a&a& Yea st I1 Yeast EAGASAELDDCRGVSENVILGQMAPIGTGAFDVMIDEE 1448 ~ouse--A-HG-S-PMK"---I1 I"--L--A"- C--LLL-A- 1478 Dros.I1 D-A-BA-T-P"""- IIM--LPXM---C--LLL-A- 1469 YeastD-AFYMXK-AVE----I11 CII---TMSI---S-K-VKGTN 1435 Yeast I K-VLDN-REQLDSP-ARIVV-KLNNV---S---LAKVP 1661 E. coli 8' --AVAGXR-EL--LK----V-RLI-A---YAYHQDRMR 1371 nuclear eukaryotic A e d gvSe i GTGFdGq consensus V G mutants rpbl-551 J J rpbl-1 F D

FIGURE6.-Amino acid sequence of intragenic suppressor mutations. The amino acid residues of homology box H of the largest RNA polymerase subunit from several organisms are aligned. The sequences of this portion of the RNA polymerase subunit are shown for mouse RNA polymerase II (AHEARNet al. 1987), Drosophila (JOKERST 1987), yeast RNA polymerase I, I1 and 111 (ALLISONel al. 1985; MEMETet a[. 1988). and E. coli (OVCHINNIKOVet al. 1982). Dashes indicate residues identical to the yeast RNA polymerase I1 sequence. The position of the last amino acid residue listed for each RNA polymerase is on the right. The consensus sequence for the nuclear eukaryotic polymerases is given with upper case letters representing completely conserved residues, and lower case letters representing residues conserved in 4 of 5 cases. The amino acid substitutions produced by the mutation rpbl-1 (NONETet al. 1987) and rpbl-551 are listed. rpbl-551, rpbl-552,and rpbl-553 are identical.

\ \ from both strains retained the original mutant phe- CL CL notypes when tested in a 226 background, indicating -33% -3 that the suppressing mutation was not linked to the original RPBl mutation(Figure 4and data not shown). Second, when the RPBl mutant centromere plasmid in the suppressed cellswas replaced with anothercentromere plasmid containing rpblAlO4, the suppressing phenotype was retained. Third, each suppressor segregated 2:2 in a cross. The s3 suppres- sor mutation exhibited a recessive, allele-specific sup- pressing phenotype; it suppressed the rpblAlOl con- ditional phenotype, but not the tighter rpblAlO3 or 12' 30' 38' rpblAlO4 conditional phenotypes. The s3 suppressor mutation was given the allele designation srb3-1 (sup- FIGURE7.-Allele specificity of the SRB2-1 mutation. Temper- I pressor of RNA polymerase B). srb3-1 was not studied aturedependent growth phenotypes of cells with different RPBl ts and cs alleles in isogenic wild-type(SRBP) and SRB2-1 backgrounds further dueto its weak suppressing phenotype.Analy- were assayed by spot testing on YPD medium and incubating at sis of diploids heterozygous for the s45 suppressor 12". SO", and 38". Strains with wild-type backgrounds are deriva- mutation demonstrated that the s45 suppressor ex- tives of 226, andstrains with SRB2-1 background are derivatives of N422. All strains are listed in Table 3. hibits a semi-dominant suppressing phenotype. This suppressor mutation was given the allele designation were identified asT to G transversions at bp 4593 (bp SRBB 1. numbering of ALLISONet al. 1985), changing amino SRB2-1, an allele-specific suppressorof CTR mu- acid 1428 of RPBl from valine to phenylalanine (Fig- tations: The ability of SRB2-1 to suppress the pheno- ure 6). sl7 and s43 areindependent isolates since they types of a variety of RNA polymerase I1 mutations are suppressors of two different heptapeptide repeat was investigated. SRB2-1 suppresses the inositol aux- mutations. s43 and s49 are also independent isolates otrophy as well as the conditional phenotypes con- (see MATERIALS AND METHODS). The suppressor mu- ferred by rpblAl01,rpblAl03, or rpblAlO4 (not tations lie in a region of the largest subunit of RNA shown). The ability of SRB2-1 to suppress other rpbl polymerase 11, denoted homology box H (JOKERST mutations was assayed by replacing the rpblAlO4 1987), whose amino acid sequence is conserved among mutation in isogenic SRB2-1 and SRB2 strains with six RNA polymerases from many organisms (Figure 6). different RPBl mutant alleles (Figure 7). Two of these Characterization of the extragenic suppressors: alleles produce cells that are cs and ts (rpbl-4, rpbl- Two suppressor isolates, s3and s45, were demon- 14) and fourof the alleles confer ts phenotypes (rpbl- strated to contain extragenic suppressing mutations 5, -6, -10, -11). All six of these alleles are single point by three criteria. First, RPBl mutant plasmids isolated mutations that map outside of the carboxyl-terminal 720 M. L. Ninet and R. A. Young .1 kb N E H EH SHHEH E NNCTCECTB B SN S I I-. I I II I1 I 11m I I1 I I I1 Functional - - ' VIII )111( SRB2- 1 dQne -PUT2 SRBZ- 1 + pCT 1 - pCT 1 1 + pCT 12 + PCT 13 - pCT 14 + pCT 2 2 FIGURE 8.-Restriction map of the SRBZ region. A restriction map of the SRBZIPUTZ genomic region of chromosome Vlll of S. cereuisiae is represented at the top of the figure. The horizontal lines below represent insert DNA of the YCp50 clone pCTl and deletion derivatives of pCTl described in Table 3. SRBZ-1 function was assayed by the ability of C3 rpblAlO3 mutant cells containing each of the plasmids to grow at 12". Thehorizontal arrow indicates the location of the PUT2 transcriptional unit. The barred horizontal line delineates the maximal boundaries of the SRB2 locus as defined with the deletion derivatives of pCTl. (B) BamHl, (T) BstEII, (E) EcoRI, (H) HindIIl, (N)NcoI, (S) SacI, (C) SaclI. repeat (C. Scafe, C. Martin, M. L. Nonet, S. Okamura strain, and allowing the cell to gap repair the missing and R. A. Young, unpublished results). Cells contain- 10 kb region encompassing SRBl with chromosomal ing SRB2-1 alone exhibitwild-type growth phenotypes sequences. The SRB2 wild-type plasmid clone pCT 19 at all temperatures and are prototrophic. At 12"C, had a restriction map identical to that of the SRB2-1 SRB2-I suppresses the cs phenotype of the rpblA104 mutant plasmid. The pCTl9 clone did not suppress mutation andthe rpbl-14 mutation,but does not the rpblAl03 or rpblAlO4 heptapeptide deletion mu- suppress the weaker rpbl-4 mutation.At 38",the tations.Southern analysis of genomic DNA from SRB2-1 mutation suppresses the ts phenotype of only SRB2-1 and SRB2 strainsdemonstrated that pCTl the rpblA104 mutation. Thus, with exception of the and pCT 19 are unrearrangedclones of the genomic cs phenotype of the rpbl-14 mutation, the SRB2-1 SRB2 region. mutation specifically suppresses the conditional and The SRB2 gene was localized to chromosome VI11 auxotrophic phenotypes of deletion mutations in the by probing a Southern blot ofyeast chromosomes heptapeptide repeat domain. Lastly, the lethal phe- separated by orthogonal-field-alternating gel electro- notype of the rpblAl31 CTR deletion mutation (g3" phoresis with pCTl insert DNA. A literature search repeats, see Table 1) in an SRB2 background, is mod- revealed that therestriction mapof pCTl insert DNA erated to a conditional viable phenotype (cs, ts, Ino-, is identical to that obtained for DNA adjacentthe slow growth) in an SRB2-1 background. PUT2 gene (BRANDRISS1983). Together,two lines of Cloning and Characterizationof SRB2: The ability evidence confirm that the molecularly defined SRB2 of the SRB2-1 mutation to suppress the cold-sensitive gene is the same as the genetically defined SRB2 locus. phenotype of a CTR deletion mutant was exploited First, the pCTl clone complements the put2-57 and to clone the SRB2-1 suppressor. A YCp50 genomic put2::HZS3mutations. Second, the SRB2-1 mutation DNA library was made from the s45 SRB2-1 suppres- is tightly linked to put2 (16 PD:O TT:O NPD). Thus, sor strainand transformed into the C3strain. Of 8000 both the genetically defined SRBP-I mutation and the primary (Ura') transformants, two transformants molecularly defined SRB2-1 clone map very close to were able to grow at 12". YCp50 genomic plasmid put2 on chromosome VZZZ. clones pCTl and pCT2were isolated from these two Phenotype of an SRB2 deletion: Northern analysis transformants, restriction mapped, and found tocon- of total and poly(A+) RNAdemonstrated that a tain the same 15 kb insert DNA (Figure 8). The SRB2- poly(A+) enriched messenger RNA of approximately 1 mutant gene was localized to a 1.9 kb EcoRI-BstEII 750 bases is the major transcript derived from the restrictionfragment by assaying forthe ability of 1.9-kb EcoRI-BstEII genomic fragmentcontaining various deletion derivatives of pCTl to suppress the SRB2. A 550 bp NcoI fragment in the middle of the cs phenotype of the rpblAlO3 mutation (Figure 8). EcoRI-BstEII fragment hybridized tothe 750 base The wild-type SRB2 gene was cloned by gene con- transcript, indicating that a portionof the SRB2 gene verting an SRBP-I deletion clone to wild-type in vivo. is encoded on this fragment. A partial deletionof the The clone was obtained by linearizing the pCTl1 SRB2 gene (srb2AlO)was created in vitro by replacing plasmid (Figure 8) with Sad, transforming a wild-type the NcoI fragment of pCT27 (Table 3)with the URA3 RNA Polymerase I1 CTR Suppressors 72 1 TABLE 3 List of strains and plasmids

Strain Alias Strain Genotype Source 222 DBY 1827, N114 MATa ura3-52 his3A200 leu2-3,112 D. BOTSTEIN 226 N247 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRPI 121 This work 227 N249 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl14] This work c1 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPI-1011 This work c3 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl-I03] This work C6 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HISJ [pRP1-104] This work v2 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPI-I07] This work v3 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-108] This work v4 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-109] This work v5 MATa ura3-52 his3A200 leu2-?,112 rpbIA187::HIS3 [pRPI-I 101 This work v7 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRPl-I 1 I] This work V8 MATa ura332 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-112] This work N15 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRPI-I31, pRPl121 This work N16 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-132, pRPl121 This work N56 MATa ura3-52 his3A200 teu2-3,112 rpblAI87::HIS3 [pRPl-140, pRPlIP] This work N398 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPI-lOIU] This work N399 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl-l03U] This work N400 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-104U] This work N418 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRPI 121 This work N422 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 SRB2-I [pRPl12] This work N447 8-5-4A MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRPI-103] This work N449 8-5-4B MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRBP-I [pRPI-103] This work N450 8-5-4c MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-I [pRP1-103] This work N451 8-5-4D MATa ura3-52 his3A200 leu2-3,I 12 rpbIA187::HIS3[pRPl-1031 This work N452 8-10 MATa ura3-52 his3A200 leu2-3,l I2 ade2 lys2A201 This work MATa ura3-52 his3A200 leu2-3,112 ade2 lys2A201 MB 331-17A MATa ura3-52 trpl put2-57 M . BRANDISS MB668-6D MATa ura3-52 his3A200 put2::HIS3 M. BRANDISS N455 8-1 1 MATa ura3-52 his3A200 leu2-3,112 ade2 lys2A2OI SRB2 This work MATa ura3-52 his3A200 leu2-3,112 ade2 lys2A201 srbPAIO::URA3 N460 8-1 1-7A MATa ura3-52 his3A200 leu2-3,112 ade2 lys2A201 This work N46 1 8- 1 1 -7B MATa ura3-52 his3A200 leu2-3,I 12 ade2 lys2A201 This work N462 8-ll-7C MATa ura3-52 his3A200 leu2-3,1 I2 ade2 lys2A201 sr62AlO::URA3 This work N463 8-1 1-7D MATa ura3-52 his3A200 leu2-3,112 ade2 lys2A201 srb2AIO::URA3 This work N487 1-4 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRPI-4] This work N488 1-5 MAT@ura3-52 his3A200 leu2-3,112 rpblAl87::HIS3[pRPI-5] This work N489 1-6 MATa ura3-52 his3A200 leu2-3,I 12 rpbIA187::HIS3 [pRPI-61 This work N492 1-10 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HlS3 [pRPl-IO] This work N493 1-1 1 MATO ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl-1 I] This work N494 1-14 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl-14] This work N496 42211-4 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-1 [pRPI-4] This work N497 42211-5 MATa ura3-52 his3A200 leu2-3,I 12 rpbIA187::HIS3 SRB2-I [pRPl-51 This work N498 42211-6 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 SRB2-I [pRPI-6] This work N50 1 42211-10 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-I [pRPI-IO] This work N502 42211-1 1 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-1 [pRPl-I11 This work N503 42211-14 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-I [pRPl-14] This work N505 4221pRP114 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3SRB2-I [pRPl14] This work s3 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3srb3-1 [pRPI-1011 This work s5 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPI-1531 This work s9 MAT@ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRP1-154] This work s17 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRP1-551] This work s2 1 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRP1-155] This work s27 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3 [pRPl-1561 This work s3 1 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3 [pRP1-157] This work s33 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRP1-158] This work s37 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRPI-I591 This work gene. The partial deletion removes approximately creating a heterozygous diploid SRB2lsrb2AlO 70% of the carboxyl-terminal SRB2 coding sequence (N455). To determine the phenotype of the SRB2 (Nonet and Young, unpublished). The deletion was deletion, the diploid was sporulated and subject to inserted into the chromosome of the diploid N452, tetrad analysis (Figure 9A). A slow growth phenotype 722 M. L. Nonet and R. A. Young TABLE 3-Continued

Strain Alias Genotype Source

s39 MATa ura3-52 his3A200 leu2-3,112 rpblAl87::HIS3[pRP1-160] This work s4 1 MATa ura3-52 his3A200 leu2-3,112 rpbIA187::HIS3[pRPI-161] This work s43 MATa ura3-52 his3A200 leu2-3,112 rpbIA187:HIS3[pRP1-553] This work s45 MATa ura3-52 his3A200 leu2-3,112 rpbIA187:HIS3 SRB2-1[pRPl-1041 This work s49 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3[pRP1-553] This work s5 1 MATa ura3-52 his3A200 leu2-3,112 rpblA187::HIS3[pRP1-162] This work YCp50 URA3 centromere yeast/E. coli shuttle plasmid M. ROSE pSB32 LEU2 centromere yeast/E. coli shuttle plasmid J. TRUEHEART pRPll2 URA3 RPBl YCp50 based centromere plasmid This work pRP114 LEU2 RPBl pSB32 based centromere plasmid This work pRP1-I URA3 rpbl-1 YCp50 based centromere plasmid This work pRP 1-4 LEU2 rpbl-4 pRPll4 based centromere plasmid This work pRP 1-5 LEU2 rpbl-5 pRPl14 based centromere plasmid This work PRP 1-6 LEU2 rpbl-6 pRPl14 based centromere plasmid This work pRP1-10 LEU2 rpbl-10 pRPll4 based centromere plasmid This work PRPI-11 LEU2 rpbl-11 pRP114 based centromere plasmid This work pRP1-14 LEU2 rpbl-14 pRPll4 based centromere plasmid This work pRP1-101 PC 1 LEU2 rpblAlOl pRPl14 based centromere plasmid This work pRP1-103 PC3 LEU2 rpblAlO3 pRPll4 based centromere plasmid This work pRP1-104 PC6 LEU2 rpblAlO4 pRPll4 based centromere plasmid This work pRP1-1OlU pClU URA3 rpblAlOl pRPll2 based centromere plasmid This work pRP1-lO3U pc3u URA3 rpblAlO3pRPll2 based centromere plasmid This work pRPI-104U pC6U URA3 rpblAlO4pRPll2 based centromere plasmid This work pRP1-107 PV2 LEU2 rpblAlO7 pRPl14 based centromere plasmid This work pRP1-108 PV3 LEU2 rpblAlO8 pRPll4 based centromere plasmid This work PRPI-109 PV4 LEU2 rpblAlO9 pRPl14 based centromere plasmid This work pRP1-I 10 PV5 LEU2 rpblAl10 pRPl14 based centromere plasmid This work pRP1-I 11 PV7 LEU2 rpblAllI pRPll4 based centromere plasmid This work pRPI-112 PV8 LEU2 rpblAll2pRPl14 based centromere plasmid This work pRP1-131 pN15 LEU2 rpblAl31 pRPll4 based centromere plasmid This work pRP1-132 pN16 LEU2 rpblA132 pRPll4 based centromere plasmid This work pRP1-140 pN56 LEU2 rpblAl40 pRPl14 based centromere plasmid This work pRP1-153 LEU2 rpblAl53 pRPl14 based centromere plasmid This work pRP1-154 LEU2 rpblAl54 pRPll4 based centromere plasmid This work pRP1-155 LEU2 rpblA155 pRPl14 based centromere plasmid This work pRP1-156 LEU2 rpblA156 pRPll4 based centromere plasmid This work pRP1-157 LEU2 rpblA157 pRPll4 based centromere plasmid This work pRP1-158 LEU2 rpblAl58 pRPll4 based centromere plaspid This work pRP1-159 LEU2 rpbl A159pRP 1 14 based centromere plasmid This work pRP1-160 LEU2 rpbIAl60 pRPl14 based centromere plasmid This work pRP1-I61 LEU2 rpblAl61 pRPll4 based centromere plasmid This work pRP1-162 LEU2 rpblA162 pRPll4 based centromere plasmid This work pRP1-551 LEU2 rpbIAlOl,551 pRPll4 based centromere plasmid This work pRP1-552 LEU2 rpblA104,552 pRPl14 based centromere plasmid This work pRP1-553 LEU2 rpblAlO4,553 pRPl14 based centromere plasmid This work pCT 1 14.6-kb genomic SRB2-1, PUT2 fragment inserted in YCp50 at BamHI This work pCT 1 1 pCT 1 derivative with two (10 kb in total) adjacent Sac1 fragments deleted This work pCTl2 pCTl derivative with 0.8-kb BamHI fragment deleted This work pCT 13 pCTl derivative with 0.8-kb BstEll fragment deleted This work pCT 14 pCTl derivative with two (1.7 kb in total) adjacent Sac11 fragments deleted This work pCT 19 14.6-kb genomic SRB2 PUT2 fragment inserted in YCp50 at BamHI This work pCT22 2.5-kb EcoRI SRBZ-1 fragment inserted in YCp50 This work pCT27 2.5-kb EcoRI SRB2 fragment inserted in pBluescript This work pCT3O 0.5-kb NcoI fragment of pCT27 replaced with 1. I-kb URA3 fragment This work segregates 2:2 in this cross. The Ura+ phenotype respectively, in SC mediumsupplemented with 100 marking the deletion segregates inall cases with the p~ inositol. Inaddition to their slow-growth pheno- slow-growth phenotype,demonstrating that the SRBB type, srb2Al0 mutants are cold-sensitive, heat-sensi- deletion produces a slow-growth phenotype. The 750 tive, and inositol auxotrophs (Figure 9B). nucleotide putative SRBB transcript is absentfrom strains carrying the srb2Al0 deletion. Growth curves DISCUSSION revealed thatthe doubling time of wild-type and We have defined three classes of mutationsthat srb2Al0 mutant cells is 110 minutes and 280 minutes, suppress the cold-sensitivity, temperature-sensitivity RNA Polymerase I1 CTR Suppressors 723 A. RPBl deletionoccurred. haveallele must Because the frequency of multipleduplications is higherthan would be expected if each duplication is an independ- ent event,it seems likely that these duplications occur via a recombination mechanism. m Three independently isolated pointmutations in RPBl were found to suppress the conditional and B. 15O 30' 37O -In0+In0 Ino- phenotypes of the rpblAIO1 and rpblAl04 CTR deletion alleles. All three of these point mutationsare T to G transversions at nucleotide 4593 of the RPBl codingregion, indicating that this position of the RPBl coding region is a mutagenic hot spot or that only a few mutations in the RPBl coding region are " capable of suppressing mutations in the repeat do- FIGURE9.-Phenotype WII of a partial deletion of the SRB2 gene. A,Ten tetrads of the diploid N455, heterozygousfor the KGiin. The isolation of thesemutations in homology srb2AIU::URA3deletion, were dissected on YPD medium by micro- box H of RPBl suggests that this region of the manipulation. The slow growth phenotype co-segregates in these interacts withthe carboxyl terminal repeat or with and ten additional tetrads (not shown) with the Ura+ phenotype other factor(s) which associate with the repeatdomain. which marks the srb2AIU::URA3 deletion. The heterozygous dip loid N455 was created by introducing pCT27 insert DNA into the It is also possible that homo1ogy box H and the CTR chromosome of N452using the method of ROTHSTEIN(1983). and domain have partially redundant functions. confirmed by Southern analysis. B, Growth phenotypes of cells Two differentextragenic suppressing mutations from a single tetrad were assayed on YPD media at 12", 30°, and were obtained, ~-2-1 and srb3-l. srb3-l suppresses 38". and on media containing or lacking 100 PM inositol at 30°C. The genotypes of the upper two spores are SRB2 and the lower two the conditional phenotype Of rpb1A1019 but not the spores are srb2AIU::URA3. The four strains are, from top to bot- tighterconditional phenotypes of rpblAlO3 and tom, N460,N461, N462 andN463 (Table 3). rpblAl04. contrast,In the SRB2-1 mutation sup- presses the cs, ts, and Ino- phenotypes of a number and inositol auxotrophy of RNA polymerase I1 CTR of deletion mutants in the heptapeptide domain, but domainconditional mutations. Two types of intra- does not suppress most other ts or cs mutations in genic mutations were obtained, spontaneous partial RPBl regardless of the strength of the phenotype duplications of the repeat coding region and point conferred by the allele. This allele specificity suggests mutations in a conserved segment adjacent the CTR a physical interaction between the RPBl CTR domain domain coding sequence. Extragenic mutationswhich and theSRB2 product. Furthermore, cells containing suppress CTR mutations in an allele specific manner a partial deletionof the SRB2 gene are phenotypically were also isolated. Results from analysis of the SRB2- slow growers, ts, slightly cs, and inositol auxotrophs. 1 extragenic suppressorlead us to suggest that its gene The Ino- phenotype is particularly characteristic of product interacts with the CTR domain. mutations that affect the mRNA transcription appa- Intragenic mutations were the major class of sup- ratus,as mutations in RPBl, RPB2 and RPBI fre- pressor of RNA polymerase I1 CTR domain condi- tional mutations. Most of these intragenic mutations quently produce Ino- phenotypes (C. SCAFE,M. No- were spontaneous partial duplications of the repeat NET, and R. YOUNG, unpublished results; WOYCHIK domain, which occur at a frequency of greater than and YOUNG 1989; ARNDT,STYLES and FINK1989). lo". The acquisition of this class of suppressors con- SRB2 is required for normal growth,SRB2-1 allele- firms that only 13 repeat unitsare needed in the CTR specifically suppresses mutations in the heptapeptide domain forwild-type yeastRNA polymerase I1 activity repeat domain of RPB1, and srblAl0 has phenotypes in vivo. The spontaneous partial duplication of the previously associated with defects in components of repeat domain could occur by two possible mecha- the transcriptionapparatus. These criteria strongly nisms. First, partial deletions or duplications of the suggest that SRB2 encodes a component of the tran- repeat domain could be created during DNA replica- scription apparatus, and that the SRBP gene product tion by melting and subsequent misannealing of the is an excellent candidate to interact with the hepta- newly replicated strand to homologous, but non-iden- peptide repeat domain. Biochemical analysis of the tical repeat units. Alternatively, recombination be- SRB2 gene productshould help elucidateSRB2's role tween homologous, but nonidentical,repeat units in transcription, and should provide important clues could account for the observed partial duplications. to the function of the RNA polymerase I1 CTR do- However, to account for the structure of the CTR main. domain of two of the suppressors, s9 and s51, two We thank C. CARPENTERpreparation for of the manuscript. This independent mis-replication events or recombination workwas supported by a grant (GM34365) from the National events between three plasmid-borne copies of an Institutes of Health. 724 M. L. Nonet and R. A. Young

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