Copyright 0 1990 by the Genetics Societyof America retl-1, a Yeast Mutant Affecting Transcription Termination by RNA Polymerase I11 Philip Jamesand Benjamin D. Hall Department of Genetics, SK-50,University of Washington, Seattle, WA 98195 Manuscript received December 8, 1989 Accepted for publication February 27, 1990 ABSTRACT In eukaryotes, extended tractsof T residues are known to signal the termination of RNA polymerase 111 transcription. However, it is not understood how the transcription complex interacts with this signal. We have developed a selection system in yeast that uses ochre suppressors weakenedby altered transcription termination signals to identify mutations in the proteins involved in termination of transcription by RNA polymerase 111. Over 7600 suppression-plus yeast mutants were selected and screened, leading to the identification of one whose effect is mediated transcriptionally. The retl-1 mutation arose in conjunction withmultiple rare events, including uninduced sporulation, gene amplification, and mutation.In vitro transcription extracts from retl-1 cells terminate less efficiently at weak transcriptiontermination signals than thosefrom RET1 cells,using a varietyof tRNA templates. In vivo this reduced termination efficiency can lead to either an increase or a further decrease in suppressor strength, depending on the location of the altered terminationsignal present in the suppressor tRNA gene. Fractionationof in vitro transcription extracts and purificationof RNA polymerase I11 has shown that the mutant effect is mediated byhighly purified polymerase in a reconstituted system. N eukaryotic cells, RNA polymerase I11 is respon- imum of six is required (ALLISONand HALL 1985). I sible forthe transcriptionof tRNA genes, the Sequences surrounding the T tract also affect termi- genes for 5s RNA, and those for a number of other nation (BOGENHAGENand BROWN198 1 ; MAZABRAUD low molecular weight RNAs of the nucleus and cyto- et al. 1987), however no consistent pattern for these plasm (for review see CILIBERTO,CASTAGNOLI and context effects has been discerned, and the lengthof CORTESE1983). The regulatory regions and the pro- the T tract is theprimary known determinant of tein factors required for initiation of transcription of terminator strength. those genes by RNA polymerase I11 have been exten- Studiesof the proteins required for termination sively studied (ALLISON,GOH and HALL 1983;LASSAR, have suggested both factor-dependent and factor-in- MARTINand ROEDER1983; GEIDUSCHEKand Toc- dependent mechanisms for RNApolymerase I11 tran- CHINI-VALENTINI1988). In tRNA and 5s genes, the scription termination. In experiments with Xenopus promoter elements governing transcription initiation Zaevis and with calf thymusRNA polymerase 111, consist primarily of intragenic sequences (SAKONJU, quasirandom transcription initiation was obtained in BOCENHAGENand BROWN1980; BOGENHAGEN, SAK- the absence of accessory transcription factors, at vec- ONJU and BROWN1980; HOFSTETTER,KRESSMANN tor DNA sites by the former enzyme and at double- and BIRNSTIEL1981). Proteins required for the tran- stranded DNA ends by the latter (COZZARELLIet al. scription of these genes include RNA polymerase I11 1983; WATSON,CHANDLER and GRALLA 1984). In as well as the transcription factors TFIIIB, TFIIIC, both cases, the resultingtranscription events were and, for the5s RNA genes, TFIIIA (SEGALL,MATSUI efficiently terminated at a downstream cluster of T and ROEDER1980; ENGELKEet al. 1980; KLEKAMP residues. From these data, it would appear that the and WEIL 1982). RNA polymerase molecule alone is sufficient for ac- Discrete transcriptformation requires that both curate transcription termination. initiation and termination occur at defined locations A different view of the polymerase 111 termination on the DNA template. However, the elements that process is derived from in vitro transcription reactions control RNA polymerase I11 transcript termination carried out in the presence of La, a protein in verte- have been characterizedonly partially. The consensus brate nuclei that binds specifically to small RNAs sequence required for termination by RNA polymer- having three or more 3”terminal uridylate residues ase I11 is a series of T residues in the noncoding strand (STEFANO1984). The activity of HeLa cell extracts of the DNA (BOGENHAGENand BROWN1981). In for in vitro transcription of pol 111 templates is affected higher eukaryotes, at least four T residues are re- in two ways by immunodepletion of the La protein. quired for efficient termination, while in yeast a min- There is both a substantial decrease in the number of Genetics 125 293-303 (June, 1990) 294 P.D. James and B. Hall pol I11 transcripts accumulated and a shortening at in tetrad analysis. The primary strains used for phenotypic the 3’ end by one or two U residues, as compared to analysis were diploids PJ2 1 and PJ26 and haploids PJ19- 33B, PJ21-47D, and PJ26-6C. Strains PJ17-1A and PJ17- pol I11 products transcribed in the presence of La. 6A were used in mating type tests. In all strains, suppressor When purified La protein is added back to the de- tRNA alleles were supplied onthe integrating plasmids pletedextracts, transcript size is restored fully but YIp5-U(IV) and YIp5-1194, described below. transcription activity is only partially restored (GOTT- Plasmids: Plasmids YIp5-U(IV) and YIp5-A94 are inte- LIEB and STEITZ1989a). These and otherobservations grating plasmids that contain a 4.5-kb fragment carrying the SUP#-U(IV) or SUP4-A94 allele inserted into the EcoRI of transcription behavior in the presence and absence and HindIII sites of the vector YIp5 (STRUHLet al. 1979). of Lahave led GOTTLIEBand STEITZ (1989b)to In YIp5-A94 the Hind111 site has been converted into an suggest that La protein is a release factor for transcrip- XhoI site. The SUP4-U(IV) and SUP4-A94 alleles have been tion termination that is required for completion of previously described (KURJAN et al. 1980; ALLISONand synthesis and for release of the nascent transcript. HALL1985). These plasmids were digested with BstEII prior totransformation in orderto target integration tothe This model can be reconciled with the previously cited genomic SUP4 locus. Plasmids used as templates for in vitro data (COZZARELLIet al. 1983; WATSON,CHANDLER transcription reactions included pDA26-94 and pDA26-96 and GRALLA1984) showing 3’ end formation by (ALLISONand HALL1985) and PTC-U(IV) andPTC-SUP4. purified RNA polymerase I11 if those apparentlycom- The latter two were derived from the vector pTC3 by the pleted pol I11 transcripts were in fact paused products insertion of SUP4 alleles carried on 266-bp AluI fragments intoa BamHI site (SHAWand OLSON1984). The SUP#- not yet released from the DNA template. AA36A37 allele was transcribed from derivativea of pBR322 The biochemical studies cited above have not pro- with the 266-bp AluI fragment inserted into the BamHI site vided a clear understanding of the factors and process (ALLISON,GOH and HALL1983). Retransformation of mu- involved in thetermination of transcription. We tant candidates was done using pCU-U(IV), pCU-G37, and wished to use an in vivo approach to identify one or pCU-A94 (pCU indicates CEN, URA3), which are derived from the PTC and pDA26 series plasmids by insertion of more gene products required forutilization of a tran- the URA3 gene into the HindIII sites, replacing the TRPl scription termination signal. To accomplish this, we gene in the process. have developed a geneticselection system in yeastthat Genetic techniques:Yeast mating, sporulation,and tetrad requiresa mutation in a trans-acting transcription analysis were carried out as described by SHERMAN,FINK protein to compensate for defects in the cognate DNA and HICKS(1 983). Standard yeast transformation was by the method ofBEGGS (1978); integrative transformation was regulatory sequences. This approach has recently accordingtoORR-WEAVER, SZOSTAKand ROTHSTEIN been used successfully inbacterial systems (GARDELLA, (1981). In vivo suppression analysis was done by first patch- MOYLEand SUSSKIND1989; SIEGELEet al. 1989; Zu- ing yeast onto YEPD plates, then replica plating after 1 day BER et al. 1989), but has not previously been applied of growth to -lys and YEPD plates. Scoring of both lysine to eukaryotes. We made use oftwo weakly suppressing auxotrophy and ade2-1 color phenotype was doneafter incubation for 3 days at 30”. alleles of the yeast SUP4 tRNAtY‘ genethat are Determination of integrated plasmid copynumber: uniquely sensitive to pol I11 termination efficiency. By Yeast DNA was prepared from strains carrying integrated providing these genes as “targets” for mutant tran- plasmids by the method of SHERMAN,FINK and HICKS scription proteins to act upon, we have been able to (1983). Approximately 2 fig of DNA was digested by a select agene mutation that altersa yeast protein restriction enzyme known to cut once within the plasmid sequence and electrophoresedon 1% agarose gels. The directly involved in transcription termination by RNA DNA was blotted by the method of SOUTHERN (1975) to a polymerase 111. While the original mutant isolate nitrocellulose membrane and was hybridized with nick- arose by multiple events, therestored suppression translated plasmid DNA, and an autoradiogram was devel- phenotype can be ascribed to a single gene mutation. oped. The presence of a plasmid-sized band is indicative of The retl-1 mutant phenotype is increased transcrip- multiple plasmid integration. Two plasmid copies us. three plasmid