Copyright 0 1992 by the Genetics Society of America

A Mutant tRNA Affects &Mediated Transcription in Saccharomyces cerarisiae Anne M.Happel' and Fred Winston Department of Genetics, Harvard Medical School, Boston, Massachusetts021 15 Manuscript received April 30, 1992 Accepted for publication June 18, 1992 ABSTRACT Mutations in the SPT3, SPT7, SPT8 and SPTIS genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by T y elements or by the T y long terminal repeat sequence, 6. These SPT genes are required for normal transcription of T y elements, and their gene products are believed to be involved in initiation of T y transcription from 6 sequences. We have isolated and analyzed extragenic suppressors of spt3 mutations. These new mutations, named rsp, partially suppress the requirement for SPTS, SPT7, SPTS and SPT15 functions. In addition, rsp mutations cause changes in transcription ofsome 6 insertions in an S P F genetic background. Interactions between mutations in the fouridentified RSP genes show a numberof interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes. Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other threeRSP genes also encode frameshift suppressor glycine tRNAs. In addition, other types offrameshift suppressor glycine tRNAs'canconfer some Rsp- phenotypes.

PT genes of Saccharomyces cerevisiae were identified by mutations that cause suppression of T y and solo 6 insertion mutations in the 5' regions of HZS4, LYS2 and URA3 (Spt = suppressor of Ty; WINSTONet al. 1984,1987; FASSLER and WINSTON1988; NATSOULIS et al. 1991). Based on mutant phenotypes and effects on transcription, four of these genes, SPT3, SPT7, SPT8 and SPTl5, comprise one class among a large group of SPT genes. This class of SPT genes is requiredforthe initiation of transcriptionfrom 6 sequences, the long terminal repeats that flank T y l and Ty2 elements (WINSTON et al., 1984,1987; EISENMANN, DOLLARD and WINSTON 1989). Several other commonphenotypes suggest that the SPT3, SPT7, SPT8 and SPTlS genes have other cellular roles in addition to a role in T y transcription. These phenotypes include the failure of homozygous diploids to sporulate, poor mating between haploid mutants, and the clumping of cells. In addition, SPT7 and SPTl5 are essential for viability (C. DOLLARD and F. WINSTON, unpublished results; EISENMANN, DOLLARD and WINSTON1989). Previous results have shown that at least spt3, spt7 and sptl5 mutations reduce transcription of mating pheromone genes (HIRSCHHORN and WINSTON1988; F. WINSTON and C. DOLLARD, unpublished results; EISENMANN et al. 1992). Biochemical, molecular, and genetic resultshave demonstrated that the SPT15 gene product, TFIID, is a general transcription factor, required for transcription initiation

S

' Present address: Department

of Molecular Biology and Microbiology,

Tufts University Medical School, Boston, Massachusetts 02 1 1 1 .

Genetics 132 361-374 (October, 1992)

of most or all genes transcribed by RNA polymerases, both in vitro and in vivo [see SAWADOGO and SENTENAC (1990), ROEDER (199 1) and SHARP(1992) for reviews]. T h e sptl5 mutants thatwe have studied are apparently defective for initiation at a subset of RNA polymerase II-dependent promoters (ARNDT et al. 1992; EISENMANN, DOLLARD and WINSTON1989; EISENMANN et al. 1992). T h e striking similarity of mutant phenotypes for spt3, spt7, spt8 and sptl5 mutants strongly suggested that the SPT3, SPT7 and SPT8 gene products are involved either with TFIID function or with some related aspect of transcription initiation. Recent work has shown that the SPTS gene product is a TFIID-associatedproteinthat is requiredfor normal TFIID function at particularpromoters in vivo (EISENMANN et al. 1992). Thisconclusion is based on several observations. First, a particular mutation in SPT15, spt15-21, that is defective for transcription of particular genes, is suppressed by specific missense mutations in SPT3. Second, the genetic interactions between sptl5 and spt3 mutations are allele specific. Third, the phenotype of an spt3 null mutant is the same as that of an sptl5-21 mutant-viable, but slow growing and defective for transcription of a number of different genes. Finally, the SPTS protein coimmunoprecipitates with theSPT15geneproduct, TFIID. Therefore, SPTS appears to be a specificity factor, required for some aspect of TFIID function for transcription of T y elements and othersequences in vivo. In the work presented here, extragenicsuppressors

A. M. Happel and F. Winston

362

of spt? mutations were isolated to identify other genes whose products may interact with, regulate, or substitute for SPT3 in recognizing transcription initiation signals. T h e results of this analysis show that these extragenic suppressors of spt? mutations, named rsp mutations (Rsp = reverses Spt- phenotype) also partially suppress spt7, spt8 and s p t l 5 mutations. In addition, rsp mutations cause changes in transcription of some 6 insertion mutations in an SP7T' genetic background. Interestingly, cloning and sequencingof one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs. Several models describing how rsp mutations may affect transcription are discussed. MATERIALS AND METHODS

Yeast strains: The S. cereoisiae strains used in this study are listed in Table 1. All strains are derived from strain S288C (MATa ga12).Since rsp mutations confer both recessive and semidominant phenotypes, we have denoted the wild-type allele with upper case letters and the mutant alleles with lower case letters. The 6 insertion mutations at HIS4, his4-9176 and his4-9126, have been described previously (FARABAUGH and FINK1980; ROEDER and FINK1982). The lys2-173R2 mutation was derived from the lys2-173 T y insertion (SIMCHEN et al. 1984). General genetic methods:Standard methods for mating, sporulation, and tetrad analysis were used (MORTIMERand HAWTHORNE 1969; SHERMAN, FINKand LAWRENCE 1978). Germination of rsp mutants was improved by dissecting tetrads immediately after sporulation and by incubating the dissection plates at 23'. All media was made as described by ROSE,WINSTONand HIETER(1990). Yeast cells were transformed by the lithium acetate method (ITOet al. 1983). Sporulation of spt3 homozygous diploids: Since spt3/ spt3 homozygous diploids do not sporulate, the following procedure was required for sporulation and tetrad analysis of these diploids. First, spt3lspt3 homozygous diploids were transformed with plasmid pFW32, which contains the SPT3 gene on ahigh copy number URA3+ plasmid (WINSTON and MINEHART1986). Second, purified transformants were sporulated by standard methods. This procedure allowed weak sporulation. Third, after tetrad dissection and growth on YPD plates, spore colonies were replica plated to plates that contain 5-fluoroorotic acid. This mediaonlyallows growth of cells that have lost the URA3+-containing plasmid (BOEKE,LACROUTE and FINK1984). Finally, the phenotypes of the progeny were scored. Isolation of mutants: To isolate rsp mutants, single colonies weregrown to saturation in 3 ml ofliquidYPD, washed twice with H20, and approximately 1-2 X lo' cells were plated on SC-Lys plates. [The parental strains are His+ and Lys- since spt3 mutations suppress the His- and Lys+ phenotypes caused by the his4-9176 and Eys2-173R2 insertion mutations (WINSTON, DURBINand FINK 1984; Figure l).] After 7-9 days of growth at 30°, Lys+ revertant colonies were replica plated to SC-Hisplates. Putative Lys+ Hiscandidates were purified on SC-Lys and replica plated to SC-His, and to YPD at 23 O , 30' and 37O . All isolates were derived from separate cultures and are therefore independent. After this initial screen, mutants were again single colony purified on selective media (SC-Lys), and then on

permissivemedia(YPD). All strains were frozen as 15% glycerol stocks and further analysis was performed on cells derived from frozen stocks. The rsp mutants were isolated from strains FW665, A13, A15 and A18 (Table 1) as spontaneous mutants (rspl-8, rsp2-66 and rsp4-27) or were isolated after mutagenesiswith UV light at 300 ergs/mm2, resulting in approximately 50% survival (rspl-57, rsp3-31, and rsp3-300). Spontaneous rsp mutations (suppressors of an spt3-101 mutation) were isolated at a frequency of approximately 1.2 X lo-'. Dominance tests: Dominance tests of the Rsp- suppression phenotype with respect to his4-9176 and his4-9126 was tested in MATa/MATa diploids by standard procedures. Dominance tests for effects on lys2-173R2 were done by mating MATa rsp spt3-101 lys2-I 73R2 his4-9176 strains to strain A120 (matal spt3-IO1 lys2-173R2 his4-9176).A matal parent was used since the Iys2-173R2 insertion is regulated by mating type (haploid lys2-173R2 strains are Lys+ while MATalMATalys2-173R2/lys2-173R2 diploids are Lys-). Since matal/MATa diploids are defective in a/a regulation (KASSIR and SIMCHEN 1976), matal/MATa 1ys2-173R2/lys2I73R2 diploids are Lys+. R N A isolation and Northern hybridization analysis: Cells for RNA isolation were grown in supplemented SD medium to 1.5-2.0 X lo7 cells/ml, and yeastRNAwas isolated as described by CARLSON and BOTSTEIN(1982). Since the reversion frequency of rsp mutations is very high, cultures for RNA preparation were inoculated to approxion mately 1.5 X lo6 withseveralsinglecoloniesgrown supplemented SD plates, and the frequency of revertants in each culture was tested by plating cells and examining the size of the colonies. RNA isolated from cultures with less than 1-2% revertants was used in experiments. Blotting and hybridization with DNA probes was performed as described (SWANSON, MALONEand WINSTON1991). RNA was crosslinked onto Genescreen (New England Nuclear) by exposure to UV radiation (1200 pW/cm2, 2 min; CHURCH and GILBERT1984). Hybridization with RNA probes was performed according to Bluescribe protocols (Stratagene Inc., San Diego). Hybridizationprobes: "P-LabeledDNA probes were prepared using a Boehringer Mannheim nick translation kit. Plasmids used as DNA probes were as follows: pFW45, a HIS4 internal BglII-Sal1 restriction fragment in pBR322 et al. 1984); B161, an internal 1.2-kb BglII re(WINSTON striction fragment from Tyl in pBR322 that hybridizes to both Tyl and to Ty2 mRNA (WINSTON et al. 1987); pFR2, containing the PYKl gene on a 3 kb Hind111 fragment; and pCC69, an ACT1 internal 1.6-kb HindIII-BamHI fragment in pBR322 ( C . CLARK and F. WINSTON,unpublished data). "P-Labeled RNA hybridization probes were made with the Bluescribesystem (Stratagene Inc., SanDiego).Plasmid pAH99 (HAPPEL,SWANSON and WINSTON1991), from which the antisense HIS4 ["PIRNA probe was made, was digested with HhaI such that transcription initiated at the to the HhaI site at +16 bp T 7 promoterandextended downstream of the HIS4 +1 of transcription. Enzymes: Restriction enzymes and T 4 DNA ligase were purchased from New England Biolabs, Inc. (Beverly, Massachusetts). Calf intestinal phosphatase was purchased from Boehringer Mannheim (Indianapolis, Indiana). Enzymes were used according to the instructions of the vendor. Construction of a genomiclibrary from an rsp4-27 mutant: Genomic DNA was isolated from strain A62 (MATa lys2-173R2 his4-9176 ura3-52 leu2-1 spt3-101 rsp4-27) and partially digested with Sau3A. DNA fragments in a size range of 10-22 kb were electroeluted from 0.5% agarose gelslices and ligated to plasmid YCp50 which had been

tRNA Effects on Transcription TABLE 1

S.cerevisiae strains A13 A15 A18 A3 1 A55 A62 A85 A109 A112 A120 A141 A143 A150 A168 A171 A178 A181 A189 A206 A210 A225 A243 A255 A269 A362 A364 A409 A422 A450 A463 A464 A470 A474 A477 A492 A495 A570 A576 A582 A587 A590 A607 A610 A61 3 FW665 FW941 FW942 FW948 FW952 FW988 FW1112 FW1227 F W 1237 FW1238 FW 1253 FW 1804 F W 1805 F W 1806 FW 1807 FY 2

S288C

MATa lys2-173R2 his4-9176 trplAl spt3-101 MATa lys2-173R2 his4-9176 spt3-101 trplAl MATa lys2-173R2 his4-9176 spt3-101 trplAl MATa lys2-173R2 his4-9176 ura3-52 rspl-57 MATa lys2-173R2 his4-9176 trplAl spt3-101 rsp4-27 ura3-52 MATa lys2-173RZ his4-9176 ura3-52 leu2-1 spt3-101 rsp4-27 MATa lys2-173RZ his4-9176 ura3-52 spt3A202 rspl-57 MATa lys2-173R2 his4-9176 ura3-52 spt3A.202 rsp3-31 MATa lys2-173R2 his4-9176 ade8 spt3-1 rspl-57 MATal lys2-173R2 his4-9176 trplAl MATa lys2-173RZ his4-9176 leu2-1 trplAl spt3-1 MATa lys2-173R2 his4-9176 trplAl spt3-1 MATa lys2-173R2 his4-9176 ura3-52 leu2-3 leu2-112 spt3-101 rsp2-66 MATa lys2-173R2 his4-9176 ura3-52 rsp2-66 MATa lys2-173R2 his4-9176 ura3-52 rsp4-27 MATa lys2-173R2 his4-9176 ura3-52 rsp3-31 MATa lys2-173R2 his4-9176 ura3-52 leu2-3 leu2-112 rsp3-31 spt3-101 MATa lys2-173R2 his4-9176 trplA1 ura3-52 rsp4-27 spt3-101 MATa lys2-173R2 his4-9126 ura3-52 leu2-1 rsp2-66 MATa lys2-173R2 his4-9176 ura3-52 trplAl rsp2-66 spt3A202 MATa lys2-173R2 his4-9176 rspl-57 spt7-217 MATa lys2-173R2 his4-9176 rsp3-31 spt7-217 MATa lys2-173R2 his4-9176 trpl A1 rsp3-31 spt8-113 MATa lys2-173R2 his4-9176 ura3-52 rspl-57 spt8-113 MATa lys2-173R2 his4-9176 ura3-52 rsp2-66 spt3-1 MATa lys2-173R2 his4-9176 leu2-1 rsp3-31 spt3-1 MATa lys2-173R2 his4-9126 leu2-1 rspl-57 MATa his4-9126 ura3-52 rsp3-31 MATa his4-9126 leu2-3 rsp4-27 MATa lys2-1286 his4-9126 leu2-3 ura3-52 rsp4-27 MATa his4-9126 leu2-3 ura3-52 rsp4-27 MATa lys2-173R2 his4-9176 trpl ura3-52 spt3A202 rsp4-27 MATa lys2-173R2 his4-9176 spt8-113 rsp4-27 trplAl MATa lys2-173R2 his4-9176 spt7-217 rsp4-27 trplAl MATa lys2-173R2 his4-9126 leu2-3 ura3-52 rsp4-27 MATa his4-9126 leu2-3 SUF15-2 MATa lys2-173RZ his4-9176 ura3-52 trplAl spt7-217 rsp2-66 MATa lys2-173R2 his4-9176 ura3-52 trplAl spt8-113 rsp2-66 MATa lys2-173R2 his4-9176 leu2-1 spt3-1 rsp4-27 MATa his4-9126 lys2-1286 trplA1 ade2-1 ura3-52 radZ-1 MATa lys2-173R.2 his4-9176 leu2-1 spt3A203::TRPl MATa lys2-173R2 his4-9176 ura3-52 trplAl rsp4-27 spt3A203::TRPl MATa lys2-173R2 his4-9176 ura3-52 trplAl rsp2-66 spt3A203::TRPl MATa lys2-173R2 his4-9176 ura3-52 trplAl rspl-57 spt3A203::TRPl MATa lys2-173R2 his4-9176 spt3-101 ura3-52 MATa his4-9176 lys2-173R2 ura3-52 MATa his4-9176 lys2-173R2 ura3-52 MATa spt3A.202 his4-9176 lys2-173R2 ura3-52 MATa his4-9176 lys2-173RZ ura3-52 spt8-113 MATa his4-9176 lys2-173RZ ura3-52 trplAl spt3-101 MATa lys2-173R2 his4-9176 ura3-52 spt7-217 MATa his4-9126 ura3-52 lys2-1286 MATa lys2-1286 ura3-52 his4-9126 MATa his4-9126 lys2-1286 ura3-52 MATa his4-9176 lys2-173R2 trplAl spt15-21 MATa his4-9176 lys2-173R2 spt15-21 rspl-57 MATa his4-9176 lys2-173R2 spt15-21 rsp2-66 MATa his4-9176 lys2-173R2 sptl5-21 rsp3-31 MATa his4-9176 lys2-173R2 spt15-21 rsp4-27 MATa ura3-52 MATa gal2 mall-1

363

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A. M. Happel and F. Winston

linearized with BamHI and then treated with alkaline phosphatase. Ligated plasmids were transformed into Escherichia coli strain HBlOl and approximately 80% of the plasmids isolated from individual HB 101 transformants contained large DNA inserts. Transformants were pooled into three sets that contained approximately 2500, 2740 and 3790 transformants. A sample of bacterial transformants from each pool was frozen as a 50% glycerol stock and plasmid DNA was also isolated from each pool and used for transformation experiments. Cloning of RSP4+: DNA adjacent to the mutantrsp4-27 allele was used to clone the RSP4+ allele by the integration and excision method (WINSTON, CHUMLEY and FINK1983). Plasmid pAHl1 is a YIp5 derivative that contains a 5.6-kb BamHI-Hind111 fragment that lies adjacent to the rsp4-27 locus. pAH1 1 was cut at asingle XhoI site within the BamHIHind111 fragment and integrated into strain FY2. DNA was isolated from stable integrants, digested with ClaI, ligated under dilute conditions, and then transformed into E. coli strain HBl 01 to recover RSPI' DNA in YIp5. DNA sequence analysis: T o sequence the rsp4-27 gene, a BamHI-BglI1 fragment was subcloned into the BamHI site of mp18 (NORRANDER, KEMPE and MFSSING 1983) in both orientations. Single stranded DNAwas isolated and seNICKquenced according to thedideoxy method of SANGER, LEN and COULSON(1977). The rsp4-27 glycine tRNA coding region was sequenced on both strands. T o sequence RSP4+, a 1.O-kb ClaI-BglII restriction fragment that contains RSP4+ was isolated and subcloned into AccI-BamHI-digested pUCI9 to create pFW258. RSP4+ DNA was sequenced by the same method, except using the double-stranded template. Genetic mapping of RSP4 RSP4 was localized to chromosome VI1 by field inversion gel analysis (CARLE,FRANK and OISON 1986). Blotting and hybridization were as deMALONEand WINSTON 1991). Genetic scribed (SWANSON, linkage of RSP4 to the RAD2 and SUFI5 genes was determined by crossing a rad2 his4-9126 strain (A587) and a SUF15-2his4-9126 strain (A495) by a rsp4-27 his4-9126 strain (A492 or A464). Progeny that contain rsp mutations were identified by scoring His phenotypes at 37 ". (At 37". rsp4-27 his4-9126 strains are His- and RSP4+his4-9126 strains are His+".) Plasmid constructions: Plasmidsused for subcloning were YCp50 (JOHNSTON and DAVIS1984)and pCGS42 (provided by Collaborative Research). The SUF16, SUF162, SUF16-3 and SUF16-4 genes were isolated as 0.55-kb HindIIISalI fragments from pR62-2, pR617-1, pR615-1 and pR628, respectively (CABER and CULBERTSON 1984). These fragments were cloned into the HindIIISalI sites of YCp50 and pCGS42. T o construct the spt3A203::TRPI allele, previously described BamHI linker insertion mutations in the SPT3 gene (WINSTONand MINEHART 1986) were utilized. The BamHI insertion spt3-374 is located near the 5' end of t h e SPT3 coding region, and the RamHI insertion spt3-382 is located 175 bp from the 3' end of the SPT3 coding region. The SPT3 sequences between these two BamHI insertions were deleted by combining EcoRIBamHI fragments from plasmids pFW32-374 and pFW32382 (WINSTONand MINEHART 1986) to produce plasmid pAH100. The TRPI gene was inserted at the site of the deletion by cloning a BamHI-BglII TRPI fragment from et al. 1979) into the BamHI site of the spt3 YRp7 (STRUHL deletion. This allele was designated spt3A203::TRPI. RSP+ Construction of spt3A203::TRPI strains: An spt3A203::TRPl strain was constructed by transforming a MATa/MATa spt3-IOI/SPT3+trplAl/trplAl diploid with a 2.25-kb Ssp1 fragment, isolated from pAH 100, thatcontains

his49 176 AT0 UAS TATA

1 ~ ~173R2 2UAS TATA

-

aenotvDe SPW spa-101

L LYS+ Lyr

FIGUREI.-Structure of the his4-9176 and lys2-173R2 mutations. Transcription and suppression phenotypes of these insertion alleles are diagrammed in SPT3+ and spt3-101 backgrounds. (Top) The 91 76 insertion is 9 bp upstream of the HIS4 + I of transcription. An SPT3+ strain produces a nonfunctional transcript that initiates in the 9176 sequence (K. ARNDT andF. WINSTON,unpublished). spf3 mutants produce a wild-type length HIS4 transcript, resulting in a His+ phenotype. (Bottom) The Iys2-173R2 insertion is within the coding region of the LYS2 gene, but the exact position has not been established (A. M. HAPPELand F. WINSTON,unpublished results). The size of the LYS2 mRNA in a lys2-I 73R2 SPT3+ strain is consistent with transcription initiating in the 3' 6 sequence of lys2-173R2 andproceeding through LYS2. In spt3 mutants, a shorter, presumably nonfunctional LYS2 mRNA is produced suggesting that transcription fails to initiate in the 3' 6 sequence of lys2-173R2. UAS, upstream activation sequence; I, transcription initiation site; the arrows indicate the direction of transcription.

the spt3A203::TRPI allele. TRPI+ diploid transformants displayed either Spt+ or Spt- mutant phenotypes; diploids that displayed Spt+ phenotypes were presumed to have arisen by gene replacement spt3-IO1 ofwith spt3A203::TRPI. When one such diploid was sporulated, all 19 tetrads thatwere analyzed displayeda 2:2 cosegregation of Trp+ and Spt- phenotypes, demonstrating that the spt3A203::TRPI allele had replaced the spt3-IO1 allele of the parental diploid. All gene replacements were verifiedby Southern hybridization analysis. RESULTS

Isolation of suppressors of spt3 mutations: To isolate suppressors of spt3 mutations, we took advantage of insertion mutations at HIS4 and LYS2 that conferoppositephenotypes.Inan SPT3+ genetic background, strains that contain his4-9176 and lys2173R2 are His- and Lys+. In spt3 mutant strains, the phenotypes caused by these two insertion mutations are reversed; thus, spt3 his4-9176 lys2-I 7 3 R 2 strains a r e His+ a n d Lys- (WINSTON,DURBINand FINK1984) (Figure 1). Suppressors of spt3 mutations wereisolated

on

tRNA Effects

from spt3 lys2-173R2his4-9176 strains by selecting for Lys+ revertants and screening for those that simultaneously acquired a His- phenotype. Suppressors were selected using three differentspt3 alleles: spt3-4, a spontaneous sPt3 allele; spt3-101, a in vitro (WIN+1 frameshiftmutationconstructed STON, DURBINand FINK1984);and spt3A202, an internal deletion that removes 696 bp of SPT3 (WINSTON and MINEHART1986). Lys+ His- revertants for each mutantwere backcrossed to anSPT3+ lys2-173R2 his4-9176 strain (Lys+ His- phenotype) to determine whether the new mutations were linked to SPT3. Six revertants of spt3-4 were examined and proved to be tightly linked to SPT3 and were assumed to be true or intragenic revertants.T h e suppressors of spt3A202 were extremely unstable, barely viable, and showed very poor sporulation and germination when crossed to an SPT3+ strain; these isolates were not analyzed further. For spt3-101, six of the ten revertant mutations were unlinked to SPT3. All further analysis was restricted to thesesix independent revertants of spt3101. These mutations were designated rsp, for Reverses Spt- Phenotype. Genetic analysis showed that allsix rsp mutants share several phenotypes. First, although rsp mutations reverse the phenotypes conferred by spt3-101 with respect to suppression of insertionmutations, they fail to suppress theother spt3-101-conferred defects, including poor mating, the inability to sporulate, and clumpiness of cells. Second, all rsp SPT3' mutants grow very slowly,with doubling times at least 1.6 times longerthan wild-type strains. Third, rsp mutations do not affect his4-9176 lys2-173R2 phenotypes in SPT3+ strains. Fourth, rsp mutants are unstable and give rise to strains with a wild-type phenotype at frequencies as high as 0.5-1 .O X lo-'. In addition, four of the six rsp mutants have aleaky temperaturesensitive-lethal phenotype on YPD plates. Finally, as described in later sections, rsp mutations partially suppress other spt3 alleles as well as mutations in other SPT genes. Rsp- phenotypes cosegregate 2:2: To verify that the Rsp- phenotypes in each mutant are conferredby a single mutation, rsp mutants were crossed by wildtype strains to examine the segregation of Rsp- phenotypes in tetrads. For eachrsp mutant strain isolated, we examined segregation of suppression of spt3-101 phenotypes (Lys+, His-) and slow growth by crossing rsp mutants by an spt3-102lys2-173R2 his4-9176 strain. Sporulation of homozygous spt3 diploids was accomplished as described in Materials and Methods. Tetrad analysis showed that, in every case, suppression of spt3-101 and the slow growth phenotype cosegregated 2:2. rsp mutations also alter the phenotypeof another 6 insertionmutation, his4-9126, in an SPT3+, back-

Transcription

365 TABLE 2

rsp X rsp crosses Cross

A . rspl-57 X rspl-57 X rspl-57 X rsp2-66 X rsp2-66 X rsp3-31 X rsp3-31 X rsp4-27 X

rsp2-66 rsp3-31 rsp4-27 rsp3-31 rsp4-27 rsp3-300 rsp4-27 rsp3-300

PD

NPD

2 2 2

1 1 1

1

3 3 0 8 3

0

7 5 3

B. rspl-8 X rspl-57

8

r s p l - 8 X rsp2-66 rspl-8 X rsp3-31 rspl-S X rsp4-27

4

2 3

TT 3 5 1 1 1 0

7 1

0 3

0 5

1 4

11

9

Segregation of rsp mutations in crosses was done in two different ways, described in detail in the text. The crosses in section A were scored for effects on his4-9126 and the crosses in section B were scored for effects on the spt3-101 suppression of his4-9176. Due to the inviability of rsp double mutants, the segregation of rsp mutations was assessed both by Rsp- phenotypes and by viability. PD, parental ditype; NPD, nonparental ditype; TT, tetratype.

ground, causing a His- phenotype at 37" (described in a later section) and the segregation of this phenotype was also tested. Tetrad analysis of sporulated rsp his4-9126/RSP his4-9126 diploids showed a 2:2 segregation pattern for the Rsp- phenotypes of His- and slow growth. Dominancetests: rsp mutations were testedfor dominance for three phenotypes: effects on spt3-mediated suppression of insertion mutations, slow growth, and leaky temperature-sensitive growth. All rsp mutations wereeither semidominant or fully dominant for the reversal of spt3 suppression phenotypes. That is, diploids homozygous for his4-9176 and lys2173R2 insertions and spt3, and heterozygous for an rsp mutation were eitherweakly Lys+ His-or strongly Lys+ His-. In contrastto this phenotype, both the slow growth and temperature-sensitive Rsp- phenotypes were fully recessive. Therefore, rsp mutations confer both dominant andrecessive phenotypes. Complementation tests: Recessive rsp phenotypes were tested for complementation by mating rsp mutants to each other in all pairwise combinations, and the phenotypes of the heterozygotes were compared to diploids homozygous for each single rsp mutation. All rsp mutations failed to complement each other for the recessive slow growth and temperature sensitive phenotypes. Therefore, the six rsp mutations define one complementation group. Linkage tests:T h e complementation tests indicated that all six rsp mutations identified a single gene. T o examine linkage between the six rsp mutations, two sets of crosses were done. Surprisingly, pairwise crosses of rsp mutants demonstrated that the six rsp mutations define four linkage groups, which we have designated RSPl, RSP2, RSP3and RSP4 (Table 2). In

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A. M. Happel and F. Winston

the first set of crosses, five of the six rsp mutations were tested for linkage to each other by pairwise crosses of rsp his4-9126 strains and testing effects on his4-9126 (Table 2A). In the second set of crosses, an rspl-8spt3-101lys2-173R2his4-9176 mutant was crossed by four differentrsp spt3-I01 lys2-173R2 his49176 mutants (Table 2B). The spt3 homozygous diploids were sporulated as described in MATERIALS AND METHODS. By this analysis, r s p l - 8 was shown to be linked to rspl-57 and unlinked to the other four rsp mutations. These results demonstrate that even though the six rsp mutations define one complementation group, they represent four unlinked genes. These crosses also showed that rsp double mutants were usually inviable. Greater than 80% of tetratype tetradsandnonparentalditypetetrads showed 3:1 and 2:2 patterns of viability, respectively, while approximately 75% of parental ditype tetrads show a 4:O pattern of viability. Allele specificity between rsp mutations and different spt3 alleles: rsp mutationswere isolated as revertants of the spt3-I01 mutation, a +1 frameshift in the SPT3 coding region. T o determine whether rsp suppression was specific for spt3-I01 or if rsp mutations also suppress other spt? alleles, rspl, rsp2, rsp? and rsp4 mutations werecrossed with three otherspt? alleles to examine the effects on expression of his491 76 and lys2-I 7 3 R 2 . For these crosses we used two different deletion mutations, spt3A202 and spt3A203::TRPI, and spt3-I, a leaky allele. The results of these crosses (Table 3) show that rsp mutations suppress these three different spt3 alleles, although in a somewhat weaker fashion than they suppress spt3101. All of the rsp mutations tested alter expression of his4-9176 in spt3-I, spt3A202,and spt3A203::TRPI mutants, reversing the phenotype from His+ to His-. However, the His- phenotype of these strains became leaky after several days growth while rspspt3-101 mutants remain tightly His-. Also, rsp mutations do not reverse the lys2-I 7 3 R 2 phenotype (from Lys- to Lys+)with spt? alleles otherthan spt3-101. Thus, mutations in RSPI, RSP2, RSP3 and RSP4 strongly suppress spt3-101 and partially suppress thethree other spt? alleles tested. Northern analysis was performed to see if the rsp reversal of spt3 suppression phenotypes was due to alterations in transcription. A wild-type (SPT?+) strain that contains his4-9176 is His-, producing an aberrantly long HIS4 transcript that initiates in the 6 sequence (diagrammed in Figure 1) (WINSTON,DURBIN and FINK 1984; K. ARNDT and F. WINSTON, unpublished). This RNA is presumed to be nonfunctional for translation of HIS4 due to translation initiation and termination codons 5' to the HIS4 translation initiation codon. spt? mutations suppress the his49176 insertion phenotype causing a His+ phenotype

TABLE 3 Effects of rsp mutations in different spt3 mutants spt3 A203spt3-101 spt3A202 SPT3+ spt3-l

::TRPI

_ Mutants

R S rspl rsp2 rsp? rsp4

_

Lys

P

+ + + +

His

+

+

-

-

Lys

-

_ His

-

+ + + +

"

~

_ Lys

His

Lys

-

+ -

+ + - + + " + " NT

+

-

~

Lys

-

His

His

+ +

"

-

Suppression patterns of rsp spt3 double mutants, spt3-101 is a frameshift mutation; spt3A202 and spt?AZU?::TRPI are internal deletions: spt3-1 is a leaky allele. The different strains were scored for suppression of his4-9176 and lys2-173R2, which are present in each strain. indicates growth after oneday; - indicates no growth after two days: N T , not determined. Phenotypes were scored on minimal media lacking either lysine or histidine and compared to growth on a completely supplemented minimal plate.

+

by altering transcriptionof his4-9176 in two ways: they eliminate &-initiated transcripts and they allow wildtype length HIS4 transcripts to be produced (WINSTON, DURBIN and FINK 1984). Northern analysis demonstrates that rsp mutations reverse spt3 suppression of his4-9176 by a change in the his4-9176 transcription pattern (Figure 2). Transcription of his4-9176 in rsp-31spt3-I01 strains is identical to transcription of his4-9176 in SPT3+ RSP+ strains: wild-type levels of &initiated transcripts are present and wild-type length HIS4 transcripts are absent (Figure 2, lane 4). The rsp-31 spt3A202 double mutant, however, only partially mimics the wild-type transcriptionpattern: wild-type length HIS4 transcripts are eliminated or greatly reduced (resulting in the His- phenotype), but in contrast to rsp-31 spt3101 strains, these strainsshow no increase in the level of &initiated transcripts (Figure 2, lane 6). Identical results were also seen with rsp4-27 spt3A202 mutants (Figure 5 , lane 5 ) and with rsp2-66 spt?A202 mutants (data not shown). rsp-31 spt3-1 mutants also lack wildtype length HIS4 transcripts like rsp-?1 spt3A202 mutants (Figure 2, lane 8). Since spt3-I is a leaky spt? allele, some &initiatedtranscription is observed in both spt3-1 and rsp spt3-I strains, making it difficult to assess whether rsp mutations cause significant changes in the levelof &initiated transcription. In summary, these results demonstrate that rsp mutations drastically reduce the level of wild-type length HIS4 message at his4-9176 for all spt? mutant alleles tested, yet rsp mutations only restore high levels of transcription initiation to thehis4-9176 sequence with the spt3-I01 allele. As expectedfromtheirphenotypes, rsp mutations have little or no effect on his491 76 in an SPT3+ background (Figure 2, lane 2). Northern analysis of T y transcripts in rsp spt? double mutants also shows some allele-specific transcrip-

_

tRNA Effects on Transcription

367

hls4-917s

+

4 U

v)

+ .r

2

P

I

hls4-9/76 HIS4 d

2 7 t?

.-

c 2

8

P

I

2

? 5 B

3

'

FIGURE2.-Northern analysis of his4-9176 in rsp spt3 mutants. Total RNA was isolated from the strains indicated (left to right): FW941, A178, FW988, A181, FW948, A109, A143 and A364. T h e probe was pFW45 which contains an internal restriction fragment of HIS4. Each lane was shown to contain approximately equivalent amounts ofRNA by rehybridizing the same filter with a probe for the P Y K l gene (shown in Figure 3).

tional effects (Figure 3). As previously described, all spt3 mutations tested abolish or greatly reduce full length T y transcription (WINSTON,DURBINand FINK 1984) (Figure 3, lanes 3, 5 and 7). T h e rsp mutations completely suppress the T y transcriptional defect of spt3-lOZ mutants, such that rsp spt3-ZOZ strains produce approximately wild-type levels of full length T y transcript(Figure3,lane 4). T h e rsp-31spt3A202 mutants (Figure 3, lane 6) produce a small amount of full length T y transcripts. Northern analysis of rsp266 spt3A202 and rsp4-27 spt3A202 double mutants showed nearly identical results (a small amount offull length T y transcripts are produced, data notshown). Thus, rsp mutations completely suppress the T y transcriptional defect ofspt3-Z0Z, and partially suppresses the T y transcription defect of spt3A202 mutations. rsp mutations do not reproducibly alter T y transcription in an spt3-Z background (Figure 3, lanes 7 and 8).However, sincespt3-Z is a leaky allele, the low level of full length T y transcripts produced in spt3-Z mutants would obscure a subtle increase in full length T y transcription caused by rsp mutations. rsp mutations also do not affect T y transcription in a wild-type background (Figure 3, lane 2). Taken together, these results demonstrate that rsp

FIGURE3.-Northern analysis of T y transcription in rspspt3 mutants. Total RNA was isolated from the strains indicated (left to right): FW941, A178, FW988, A181, FW948, A109, A143 and A364. This figure shows reprobing the filter that was used inFigure 2. The probes were B161, which contains an internal restriction fragment of T y l , and and pFR2, which contains the PYKl gene.

mutations alter transcription in all spt3 mutants examined,includingan spt3 deletionmutant. Therefore, rsp mutations can partially bypass the requirement for SPT3 function in transcription of T y elements and of his4-9176. However, rsp suppression of is significantly the spt3-IOZ transcriptionaldefects stronger than is suppression of the other spt3 alleles tested with respect to &initiated transcription. This result may explain why rsp mutations reverse thelys2Z73R2 suppressionphenotype only in conjunction with the spt3-ZOZ allele, since efficient transcription initiation from the 3' 6 of lys2-Z73R2 appears to be required forLYS2 expression. rsp mutations also suppress mutations in SPT7, SPT8 and SPT15: TheSPT3,SPT7,SPT8and SPT 15 gene products are believed to act at thesame step in the initiation of transcription from6 sequences (WINSTON et al. 1987; EISENMANN, DOLLARD and WINSTON 1989). Therefore, we examined rsp spt7, rsp spt8 and rsp sptZ5 mutants to determine if rsp mutations also reverse the spt7, spt8 and sptZ5 Spt- phenotypes. Since these spt mutations strongly suppress his4-9Z76, this phenotypewas the clearest indicator of an effect of rsp mutations on spt mutations. Indeed, rspZ, rsp2, rsp3 and rsp4 mutations all reversed spt7, spt8 and sptZ5 suppression of his4-9176, changing the phenotypefrom His+ to His- (Figure 4; datanot shown).

A. M.Happel and F. Winston

368

-histidine

complete R S P 4r+s p 4 - 2 7

R S P 4r +s p 4 - 2 7 FIGURE4.-Growth phenotypes of different spt rsp double mutants with respect to suppression of his4-9176. Strains were grown on YPD plates and then replica plated to SC-His and SC plates. All plates were incubated at 30". The photographs were taken after 2 days of incubation.

SPP spt3-101 spt3A203 spt7-2 17 spt8-7 13 spt75-21

a

hiS4-9176 d HIS4 d

-4

hlr4-9176

Y)

II1-u

E

PYKl d

FIGURE 5,"Northernanalysis of his4-9176 transcription in rsp spt7 and in rsp spt8 mutants. Total RNA was isolated from strains (left to right): FW942, S288C, A171, FW948, A470, A141, A582, FW 1 1 12, A477, FW952 and A474. probes The were pFW45which contains an internal restriction fragment of HIS4 and pFR2 which contains thePYKI gene. ThePYKI transcripts shows approximately equivalent amountsof RNA in each lane.

Northern hybridization analysis of RNA isolated from rsp spt7 and rsp spt8 double mutants demonstrated that the change in phenotype corresponds to an alteration of transcription. T h e pattern of h i d 9176 transcription in the rsp4-27 spt7 and rsp4-27 spt8 doublemutants is similar to that seen in the rsp spt3A202 mutants: the level of the wild-type length HIS4 mRNA is greatly reduced, while the low level of the &initiated transcriptappears to beunaffected (Figure 5, compare lanes 5 , 9 and 1 1). Identical results were also obtained with rsp2-66 and rsp-31 mutations (data not shown). rsp mutations alter transcriptionin an S€"+ back-

ground: his49126 is a solo 6 insertion mutation that confers a cold-sensitive His- phenotype (His- at 23" , His+" at 37"). This temperature effect is caused by alterations in transcription of hid-9126 at different temperatures(HIRSCHMAN,DURBINand WINSTON 1988). To determine if rsp mutations affect his4-9126 expression, rsp hid-9126 strainswereconstructed. Analysis of these strainsshows that rsp mutations alter the phenotype of hid-9126 (in an SPTC background), such that rsp his49126 strains have a tight His- phenotype at all temperatures tested (23", 30", and 37"; data not shown). These results demonstrate that rsp mutations can alter expression of an insertion mutation even in an SPTC genetic background. Northern analysis of rsp hid-9126 mutants demonstrates that rsp mutations change the transcription of hid-9126 (Figure 6). In RSP+ strains, the previously identified, &-initiated HIS4 transcript is synthesized (SILVERMAN and FINK HIRSCHMAN, 1984; DURBIN and WINSTON1988) (Figure 6A, lane 1). In rsp mutants, however, two HIS4 transcripts are made: a minor 6initiatedtranscript and amajortranscript that is smaller than wild-type HIS4 mRNA (Figure 6A,lanes 3-6). RNase protection experiments indicate thatthis smaller HIS4 transcript corresponds to initiation approximately 105 bp downstream of the wild-type HIS4 transcription initiation site, within the HIS4 coding sequence (data notshown; diagrammed in Figure 6B). rsp mutations do not affect transcription of HZS4+: T o determine if rsp mutations might act via HIS4 promoter sequences, H I S P transcripts were examined in rsp mutants. For each rsp mutant, only wildtype length HIS4 transcripts were produced. Densitometric analysis of HIS4 transcript levels showed that rsp mutants contain 70-8096 of the wild-type level of HIS4 transcripts (data not shown). These results suggest that rsp mutations do not affect transcription of hid-9126 and his4-9176 via HIS4 sequences alone and that rsp mutations are likely to act via the 6 sequences of these loci to cause changes in transcription. Cloning and sequencing of rsp4-27: The rsp4-27

tRNA Effects on Transcription P

A

Y

his4-9126

his4-9126 -b HIS4 -b

B

BnotvDB his49126 hls4-9126 rnp

ATG

-97

+63

DhenotvDe

b

HisHis-

FIGURE6.-(Top) Northern analysis of his49126 transcription in rsp mutants. Total RNA was isolated from strains (left to right): FW1237, S288C. A409, A206, A422 and A463. All strains were grown at 30". The probe was a 52P-labeledribonucleotide probe that contained antisense HIS4 sequences from +532 to +16 nucleotides from the +I of transcription initiation (Sal1 to HhaI). Equivalent amounts of RNA were shown to be in lanes 1 , and 3-6 by hybridimtion with PYKl gene (not shown). Lane 2 contains IO-fold less RNA than allother lanes. (Bottom) A diagram of his49126 and the transcripts made in RSP and rsp strains.

mutation is dominant for reversal of spt3 insertion phenotypes and this dominant phenotype was used to clone rsp4-27. As described in MATERIALS AND METHODS, a yeastgenomic library was constructed from DNA prepared from an rsp4-27 mutant (strain A62) and wasused to transform strain FW988 (spt3-101 lys2-173R2 his4-9176 uru3-52) which is Lys- His+due his4-9176 and lys2-173R2. to spt3 suppressionof FW988 Ura+ transformants were screened for those that acquired the dominant rsp suppression phenotypes(Lys+His-).Plasmidsisolated from 36 candidates all contained an overlapping set of restriction fragments when digested withEcoRI and HindIII, suggesting thatthe same genomic DNA had been cloned in each case.

369

T o demonstrate that rsp4-27 had been cloned, a restriction fragment from the putative clones that contained rsp4-27 function was shown to direct integration of a plasmid atthe RSP4 locus. A 6.6-kb BamHI fragment was subcloned into the integrating vector YIp5 (STRUHL et al. 1979). This plasmid was digested at a single XhoI site within the 6.6-kb fragment and transformed into an R S P his4-9126 ura35 2 strain (FW 1227), selecting for Ura+ transformants. Almost all Ura+ transformants were His- at 37" and showed a slow growth phenotype, demonstrating that the putative rsp4-27 mutant gene associated with the plasmid is dominant for all Rsp-phenotypes in haploid strains. One such transformant was crossed to strain A450 (MATa his4-9126rsp4-27 leu2-3 t r p l A l ) and tetrads were dissected. Of 15 tetrads analyzed, all 15 displayed 4:O segregation for slow growth and for a His- phenotype at 37", demonstrating that the plasmid had integrated at theRSP4 locus. Southern analysis of the RSP4 locus also confirmed that the plasmid had integrated at RSP4 (data not shown). Interestingly, rsp4-27 clones confer dominant slow growth defects in a wild-typehaploid strain, even though this phenotype is recessive in an RSP4/rsp427 diploid. This result confirms that the rsp4-27 mutation is a gain-of-function mutation and explains why several attempts to clone RSP genes by complementation of the recessive slow growth and temperature sensitive defects with genomic libraries from RSP4+ strains were unsuccessful. Subcloning of the 6.6-kb BamHI fragment showed that rsp4-27 is encoded on a 450 bp BumHI-BglII DNA fragment. Surprisingly, the sequenceofthis DNA fragment showed that it contains a glycine tRNA gene, nearly identical in sequence to suf16+ (GABER and CULBERTSON 1982) and a partial u sequence (DEL REY,DONHAUE and FINK1982; SANDMEYER and OLSON 1982). No other tRNA genes or open reading frames were present on thisDNA fragment. The DNA sequence of rsp4-27 contains an additional adenine at the junction of the stem and anticodon loop. This mutation would change the anticodon of the tRNA from 3'-CCG-5' to 3'-CCGU-5', which would correct the reading frame of the spt3-101 frameshift mutation (Figure 7). Therefore, the sequence data strongly suggests that rsp4-27 encodes a novel frameshift suppressor glycine tRNA. T o verify that the glycine tRNA gene is RSP4, we also cloned and sequenced the RSP4+ gene, as described in MATERIALS AND METHODS. The RSP4+ sequence is identical to suf16+ and differs from rsp4-27 only by the single adenine. This result proves that the rsp4-27 mutation is due to asingle basepair insertion in the region of the gene corresponding to the tRNA anticodon loop. Mapping RSP4 The RSP4 gene was localized to

370

A. M. Happel and F. Winston &-on

A-OH 3'

A.

3,

C C

C C

A

A

DO-C

51

5 ' DO-c

C-Q

C-0

G-C C-0 A-0

A-U

uO-' m uQAm

QuA-

Q

ccccoo~ c u

1111

0

c "A

IIIII

0

cc

aG C-0

A-U A-U C-0

RSP4+

0.

rsp4-27

SPT3*

5 * -0AU 000 CQA CAQ QCA A 0 0 Omr ACA UOO AGQ- 3 '

Upt3-101

5'-QAU 000 CQA

= C A

rap4 -1 7

QQCA AQU nn0 ACA Omr AQQ-3'

IIII

3"CcW-5'

FIGURE7.-(A) The sequence of the tRNAs encoded by RSP4+ and by rsp4-27. The rsp4-27 mutation results in the insertion of an A (circled) in the anticodon loop. (B) The sequence of a portion of the mRNAs encoded by SPT3+ and by spt3-101. spt3-IO1 causes the insertion of 4 bases into the mRNA (marked by underlining). This frameshift mutation can be suppressed by the 4-base anticodon of the mutant rsp4-27 tRNA. TABLE 4 rsp4 linkage data Cross

NPD PD

rsp4-27 X SUF15-2 rsp4-27 X rad2 2

30 5

0

TT

cM

28 17

26 Unlinked

Mapping of rsp4-27. The data above are from crosses A492 A495 and A464 X A587. Abbreviations as in Table 2.

X

the distal portion of the right armof chromosome VZZ by hybridization of the 6.6-kb BamHI restriction fragment that contains the cloned rsp4-27 gene to a chroet al. mosomal blot of strain YP148 DNA (VOLLRATH 1988). Tetrad analysis shows that RSP4 is 26 centimorgans from SUFI5 andis unlinked to RAD2 (Table 4). Since no previously identified glycine tRNA gene maps to this region of the chromosome (MORTIMERet al. 1989), RSP4 identifies a new glycine tRNA gene. Allfour RSP genesprobablyencodeglycine tRNAs: Several pieces of evidence indicate that the four RSP genes are closely related: mutations in all four RSP genes fail to complement each other, all confer nearly identical mutant phenotypes, and rsp doublemutants are generally inviable. T o address whether the RSPl,RSP2 and RSP3 genesalso encode glycine tRNAs, a wild-type glycine tRNA gene was overexpressed in rspl, rsp2, rsp3 and rsp4 mutants. T o do this the suf16+ gene was cloned into plasmids

YCp50 (a low copy number vector) and into pCGS42 (a high copy number vector). Both plasmids were introduced intorsp his4-9126 ura3-52 strainsby transformation and Rsp- phenotypes were tested. The pCGS42-suf16+ transformants were His+ at 37" and formed colonies at nearly a wild-type rate, while the YCp5O-sufl6+ Ura+ transformants were His- at 37" and grew slowly. Thus, overexpression of suf16+ suppressed Rsp- phenotypes. This result stronglysuggests that mutations in all four RSP genes encode mutant glycine tRNAs whose effects can be diluted by the addition of many copies of a wild-type glycine tRNA. Effects of otherframeshiftsuppressorglycine tRNAs: T o address whether other frameshift mutations in glycine tRNAs would confer Rsp- phenotypes, three genesthatencode +1 frameshiftsuppressor tRNAs with different anticodons (SUFl6-2,3'-CCCC5 ' ; SUFl6-3, 3'-CCCA-5'; andSUF16-4,3'-CCCU-5': GABERand CULBERTSON 1984) were subcloned into YCp50 (low copy number) and pCGS42 (high copy number) vectors. These plasmids were thenintroduced into an S P P his4-9126 ura3-52 strain (FW 1238). YCp50-SUFI6 transformantsdid grow more slowly than wild type, but did not display Rspphenotypes. However, all of these frameshift suppressors, when present on high copy number plasmids, caused Rsp- phenotypes. For example, an S P P his#9126 strain, when transformed with a high copy number plasmid that contains SUF16-2, SUF16-3 or SUF16-4 was His- at 37' and grew slowly (Figure 8). Therefore, other glycine frameshift suppressors on high copy number plasmids mimic the phenotype of rsp4-27 on a low copy number plasmid or in itsnormal genomic location. pCGS42SUF16 frameshift suppressor plasmids were also transformed intospt3, spt7 and spt8 mutants to test whether theseplasmids affected spt suppression of his#-9176. Attempts to obtain transformantsof spt3101 and spt7-217 mutants were unsuccessful; only extremely tiny colonies were obtained. Presumably the overexpression of frameshift suppressor tRNAs causes extreme sickness in these hosts. In spt8 strains, pCGS42-SUFI6 frameshift plasmids conferred an extreme slow growthphenotype and transformants often reverted to awild-type phenotype. These transformants had Rsp- phenotypes; they reversed spt8113 suppression of his#-9176 (Figure 8). Thus, three different frameshift suppressor mutations in SUFI 6 are able to cause rsp phenotypes when expressed on high copy number plasmids. DISCUSSION

Six independent rsp mutations were isolated as extragenic suppressors of spt3-101. Although these mutations define one complementation group, they represent four unlinked RSP genes. We have shown that

tRNA Effects on Transcription A. his4-9126

transformants SC-ura

B. his4-9176

371

sptb-113

SC-ura, -his

transformants

ScUra

scum, n i s

d

FIGURE%--(A) Phenotypes of FW 1237 (his49126 S P T )transformed with high copynumber plasmids carrying the SUFI6 alleles indicated below. Suppression o f his49126 was scored at 3 7 ” . (B) Phenotypes of FW952 (spt8-I13 his4-9176) transformed with high copy number plasmids carrying the SUFI6 alleles indicated below. Suppression of his4-9176 was scored at 30”. FW1237 and FW952 were transformed with the followingplasmids: a, pCGS42SUFI6-2; b, pCGS42SUFI6-3 c, pCGS42SLIFI6-4;d, YCp50-rsp4-27; e, pCGS42-suf16+;f, pCGS42; g. YCp50. Three transformants are shown for each combination.

-.

the rsp4-27 mutation encodes a mutant glycine tRNA, and that its sequence strongly suggests that the rsp427 allele encodes a frameshift suppressor tRNA. We believe that rspl, rsp2 and rsp3 also encode frameshift suppressor glycine tRNAs, since overexpression of a wild-typeglycine tRNA gene suppresses the Rspphenotypes in rspl, rsp2, rsp3 and rsp4 mutants. Mutations in all four RSP genes alter expression of &insertion mutations in S P P , spt3,spt7,spt8 and sptl5 genetic backgrounds. In all casesthat have been examined, the change in expression correlates with an alteration in transcription. Two of the 6 insertion mutations studied are in the HIS4 promoter. However, rsp mutations did not significantly alter wildtype HIS4 transcription, suggesting that rsp mutations act (directly or indirectly) via 6 sequences to affect the transcription of these loci. Including RSP4, approximately 15 genes in the S. cereuisiue haploid genome are thought to encode glycine tRNAs with a 5’-GCC-3’ anticodon (MENDENHALL et al. 1987). The apparent instability of both chromosomal rsp mutations and rsp mutations on

plasmidsprobably occurs becausetRNAgenes are repeated. rsp mutations could revert to a wild-type phenotype by mitotic gene conversion between the mutant rsp gene and oneof the otherwild-type glycine tRNAs (SZANKASI et al. 1986). Given the poor growth of rsp mutants, any wild-type recombinants would be greatly enriched for during growth. Experiments inwhich the number of mutant or wild-type glycinetRNA genes present in both haploids and diploids was varied suggestthat thedegree of the Rsp- phenotypes is proportional to theratio of mutant to wild-type glycine tRNA present. These dosage effects may explain why mutations in different RSP genes fail to complement for recessivephenotypes (slow growth and temperature sensitive growth), and why phenotypes that are recessiveindiploids are dominant in haploids. For example, in rsp haploids, a ratio of one frameshift glycine tRNA gene to approximately fourteen wild-type glycine tRNAs genes may be responsible for producing Rsp- mutant phenotypes. Some of these phenotypes are recessive in heterozygous diploids, which would have a ratio of one

372

A. M.Happel and F. Winston

frameshift suppressor glycine tRNA gene to approximately 29 wild-type glycine tRNAs genes. Thus, the poisonous effects of the rsp mutation would be diluted approximately twofold by the products of an additional fifteen wild-type glycine tRNAs genes. Diploids heterozygous for two different rsp mutations, however, would carry two frameshift suppressor tRNA genes to approximately 28 wild-type genes, such that the ratio of frameshift to wild-type tRNAs is equal to the haploid situation. Therefore, diploids heterozygous for two rsp mutations are expected to display mutant phenotypes causing rsp mutations to fail to complement. Both genetic and transcriptional analysis of rsp spt3 mutants showed that rsp mutations partially suppressed all sPt3 alleles examined, yet conferred both stronger and morespecific effects in combination with the spt3-101 allele. Informational suppression resulting in the production of some functional SPT3 product almost certainly explains the suppression of spt3101 by rsp mutations. However, frameshift suppression at SPT3 cannot explain rsp effects on two different spt3 deletions, spt3A202 and spt3A203::TRPI, that remove greater thantwo-thirds and virtually all of the SPT3 coding sequences, respectively. Since rsp mutationscannot be informationalsuppressors of these spt3 deletion alleles, we suggest that rsp mutations can partially bypass the requirement of SPT3 function by a mechanism separate from frameshift suppression at SPT3. rsp mutations were also found to partially suppress spt7, spt8 and sptl5 mutations. Since both spt7-217 and spt8-113 mutations were isolated spontaneously, it is very unlikely that both mutations result from a specific +1 frameshiftmutationthatcould be suppressed by a glycine frameshiftsuppressortRNA. Therefore, it seems very unlikely that rsp mutations are informational suppressors of spt7-217 and spt8113 mutations. The spt15 mutation used in this study, spt15-21, has been sequenced and is known to be a et al., 1992). Since rsp missense mutation (EISENMANN effects on his4-9176 expression are similar in spt3A, spt7,spt8 and sptl5 mutants, we suggest that rsp mutations act by the same mechanism to cause these effects. Surprisingly, rsp mutations also cause changes in the transcription of the his4-9126 insertion in an SPT+ background, producing a novel nonfunctional HIS4 transcript that initiates downstream of the wild-type HIS4 transcription initiation site. It seems unlikely that rsp mutations alter his4-9126 transcription via an effect on SPT3, SPT7, SPT8 or SPT15, since neither wild-type nor mutant alleles of these genes produce this novel transcript. However, the SNF2, SNF5 and SNF6 genes are also required for T y transcription, and snj2 his4-9126 mutants produce a nonfunctional

HIS4 mRNA that initiates at an identical position as the rsp his4-9126 transcript internal to HIS4 (HAPPEL, SWANSON and WINSTON1991). Thus, one possibility is that rsp mutations affect SNF2, SNF5 or SNF6 to produce the novel HIS4 transcript. Although tRNAs are not known to be components of RNA polymerase 11-mediated transcription, we cannotexclude the possibility thatthese glycine tRNAs are directly involved in affecting transcription of some loci. In addition to their role in translation, tRNAs have been found as cofactors in many cellular processes, including reverse transcriptionof retroviral RNA (VARMUS and BROWN1989), thebiosynthesis of 6-aminolevulinate (KANNANGARA et al. 1988) and in the conjugation of ubiquitin to the acidic termini of proteins(FERBER and CIECHANOVER 1987). Yeast tRNA has also been reportedto bind yeast RNA polymerase I1 in a 1:1 ratio and toinhibit nonspecific elongation of transcription (SAWADOGO 1981). Therefore, one possible explanation of Rsp- phenotypes is that these mutant glycine tRNAs directly affect transcription of several loci, including those that contain 6 sequences. Alternatively, rsp-encoded mutant glycine tRNAs may indirectly affect transcription to cause Rsp- phenotypes. One possibility is that rsp-encoded frameshift tRNAs may alter the translation of a specific protein involved in transcription of these &insertion loci. By this model, eitherproduction of amutantframeshifted product or a reduction in the amount of this protein causes the rsp transcriptional defects. Experiments involving the SUFI6 frameshift mutations argue against this model. T h e nucleotide sequence of the anticodon of the rsp4-27 tRNA (3’-CCGU-5’) differs from SUF16-2 and SUF16-3 at both position 3 and 4 and differs from SUF16-4 at position 3. Complementary pairing of the first three nucleotides of the anticodon (including wobble base pairing at position3) shows that rsp mutations would notbe expected to recognize the same codons as the SUF16 frameshift tRNAs. Since both types of mutant glycine tRNAs produce Rsp- phenotypes, this argues that rsp mutations are not causing frameshifting of a specific protein to produce Rsp- phenotypes. In a second general model, rsp mutations may cause a change in the cell’s physiologythat results indirectly in Rsp- transcriptional phenotypes. Either frameshifting of proteins or a general translational defect may be caused by these mutant tRNAs and may invoke a general response analogous to the heatshock response found in all cells(reviewed by SCHLESINGER, ASHBURNER and TISSIERES 1982) or the stringentresponse of E. coli (GALLANT1979) and ofyeast (WARNER and GORENSTEIN 1978).Inboth cases, transcription of certain classes of genes is altered in response to an altered cellular physiology. The signal for the strin-

tRNA Effects on Transcription

gent response in yeast appears to be uncharged tRNA (WARNERand GORENSTEIN 1978). Conceivably, rsp mutations might impair aminoacylation of the mutant tRNA, resulting in uncharged tRNA in the cell, and thereby causing a change in transcription. Thus, rsp mutations(and SUF16 frameshiftsuppressorsexpressed on high copy number plasmids) could be causing translational defects that result in an altered cellular physiology that in turn alters transcription. This model fits well with our results with different SUFI6 frameshift suppressors. In these experiments Rsp- phenotypescorrelated well with sickness and slow growth. Thus, theSUFI6 low copy transformants grew more slowly than wild type, but did not display Rsp- phenotypes.High copy SUFI6 transformants grew extremely slowly and showed Rsp- phenotypes. Therefore, the SUFl6 frameshift suppressor tRNAs appear to be more toxic when overexpressed. This effect may alter the physiology of the cell and result in Rsp- phenotypes. In summary, our results demonstrate that mutant tRNAs can cause changes in transcription in vivo. It seems likely that these mutant tRNAsaffect transcription of many sequences beyond those examined in these studies. T h e simplest hypothesis is that the mutant glycine tRNAsact via one common defect to directly or indirectly cause changes in transcription that are unrelated toframeshift suppression at SPT3. We thank STEPHANIE RICUPERO for cloning and sequencing the wild-type R S P P gene. We also thank PATRICIA MINEHART, BARBARA BERGand KARENARNDTfor helpful comments on the manuscript. This work was supported by National Institute of Health grant GM32967, National Science Foundation grant DCB8451649 and a grant from the Stroh Brewery Company to F.W.

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A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae.

Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused...
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