retl-1, a yeast mutant affecting transcription termination

Copyright 0 1990 by the Genetics Society of America

retl-1, a Yeast Mutant Affecting Transcription Termination by RNA Polymerase I11

Philip James and 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 tracts of 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 weakened by 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 with multiple rare events, including uninduced sporulation, gene amplification, and mutation. In vitro transcription extracts from retl-1 cells terminate less efficiently at weak transcription termination signals than those from RET1 cells, using a variety of 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 termination signal present in the suppressor tRNA gene. Fractionation of in vitro transcription extracts and purification of RNA polymerase I11 has shown that the mutant effect is mediated by highly purified polymerase in a reconstituted system.

I N eukaryotic cells, RNA polymerase I11 is responsible for the transcription of tRNA genes, the

genes for 5s RNA, and those for a number of other low molecular weight RNAs of the nucleus and cyto- plasm (for review see CILIBERTO, CASTAGNOLI and CORTESE 1983). The regulatory regions and the protein factors required for initiation of transcription of those genes by RNA polymerase I11 have been exten- sively studied (ALLISON, GOH and HALL 1983; LASSAR, MARTIN and ROEDER 1983; GEIDUSCHEK and Toc- CHINI-VALENTINI 1988). In tRNA and 5s genes, the promoter elements governing transcription initiation consist primarily of intragenic sequences (SAKONJU, BOCENHAGEN and BROWN 1980; BOGENHAGEN, SAK- ONJU and BROWN 1980; HOFSTETTER, KRESSMANN and BIRNSTIEL 1981). Proteins required for the transcription of these genes include RNA polymerase I11 as well as the transcription factors TFIIIB, TFIIIC, and, for the 5s RNA genes, TFIIIA (SEGALL, MATSUI and ROEDER 1980; ENGELKE et al. 1980; KLEKAMP and WEIL 1982).

Discrete transcript formation requires that both initiation and termination occur at defined locations on the DNA template. However, the elements that control RNA polymerase I11 transcript termination have been characterized only partially. The consensus sequence required for termination by RNA polymerase I11 is a series of T residues in the noncoding strand of the DNA (BOGENHAGEN and BROWN 1981). In higher eukaryotes, at least four T residues are required for efficient termination, while in yeast a min-

Genetics 1 2 5 293-303 (June, 1990)

imum of six is required (ALLISON and HALL 1985). Sequences surrounding the T tract also affect termination (BOGENHAGEN and BROWN 198 1 ; MAZABRAUD et al. 1987), however no consistent pattern for these context effects has been discerned, and the length of the T tract is the primary known determinant of terminator strength.

Studies of the proteins required for termination have suggested both factor-dependent and factor-independent mechanisms for RNA polymerase I11 transcription termination. In experiments with Xenopus Zaevis and with calf thymus RNA polymerase 111, quasirandom transcription initiation was obtained in the absence of accessory transcription factors, at vector DNA sites by the former enzyme and at double- stranded DNA ends by the latter (COZZARELLI et al. 1983; WATSON, CHANDLER and GRALLA 1984). In both cases, the resulting transcription events were efficiently terminated at a downstream cluster of T residues. From these data, it would appear that the RNA polymerase molecule alone is sufficient for accurate transcription termination.

A different view of the polymerase 111 termination process is derived from in vitro transcription reactions carried out in the presence of La, a protein in verte- brate nuclei that binds specifically to small RNAs having three or more 3”terminal uridylate residues (STEFANO 1984). The activity of HeLa cell extracts for in vitro transcription of pol 111 templates is affected in two ways by immunodepletion of the La protein. There is both a substantial decrease in the number of

294 P. James and B. D. Hall

pol I11 transcripts accumulated and a shortening at the 3’ end by one or two U residues, as compared to pol I11 products transcribed in the presence of La. When purified La protein is added back to the de- pleted extracts, transcript size is restored fully but transcription activity is only partially restored (GOTT- LIEB and STEITZ 1989a). These and other observations of transcription behavior in the presence and absence of La have led GOTTLIEB and STEITZ (1989b) to suggest that La protein is a release factor for transcription termination that is required for completion of synthesis and for release of the nascent transcript. This model can be reconciled with the previously cited data (COZZARELLI et al. 1983; WATSON, CHANDLER and GRALLA 1984) showing 3’ end formation by purified RNA polymerase I11 if those apparently com- pleted pol I11 transcripts were in fact paused products not yet released from the DNA template.

The biochemical studies cited above have not provided a clear understanding of the factors and process involved in the termination of transcription. We wished to use an in vivo approach to identify one or more gene products required for utilization of a transcription termination signal. T o accomplish this, we have developed a genetic selection system in yeast that requires a mutation in a trans-acting transcription protein to compensate for defects in the cognate DNA regulatory sequences. This approach has recently been used successfully in bacterial systems (GARDELLA, MOYLE and SUSSKIND 1989; SIEGELE et al. 1989; Zu- BER et al. 1989), but has not previously been applied to eukaryotes. We made use of two weakly suppressing alleles of the yeast SUP4 tRNAtY‘ gene that are uniquely sensitive to pol I11 termination efficiency. By providing these genes as “targets” for mutant transcription proteins to act upon, we have been able to select a gene mutation that alters a yeast protein directly involved in transcription termination by RNA polymerase 111. While the original mutant isolate arose by multiple events, the restored suppression phenotype can be ascribed to a single gene mutation. The retl-1 mutant phenotype is increased transcrip- tional read-through of weak RNA polymerase I11 terminators and can be demonstrated both in vivo and in vitro. Protein fractionation and complementation of the in vitro transcription phenotype have shown that RET1 cells have a functional alteration in the RNA polymerase I11 core enzyme, indicating that the polymerase itself is involved in the in vivo recognition of and response to transcription termination signals.

MATERIALS AND METHODS

Media and strains: 5-Flouro-orotic acid plates were as described by BOEKE, LACROUTE and FINK (1984). All other media were as described by SHERMAN, FINK and HICKS (1983). Yeast strains are described in Table 1. Diploid PJ14 and haploid PJ6-3C were used for mutant selection. The PM3958 mutation was crossed into the diploid PJ2 1 for use

in tetrad analysis. The primary strains used for phenotypic analysis were diploids PJ2 1 and PJ26 and haploids PJ19- 33B, PJ21-47D, and PJ26-6C. Strains PJ17-1A and PJ17- 6A were used in mating type tests. In all strains, suppressor tRNA alleles were supplied on the integrating plasmids YIp5-U(IV) and YIp5-1194, described below.

Plasmids: Plasmids YIp5-U(IV) and YIp5-A94 are integrating plasmids that contain a 4.5-kb fragment carrying the SUP#-U(IV) or SUP4-A94 allele inserted into the EcoRI and HindIII sites of the vector YIp5 (STRUHL et al. 1979). In YIp5-A94 the Hind111 site has been converted into an XhoI site. The SUP4-U(IV) and SUP4-A94 alleles have been previously described (KURJAN et al. 1980; ALLISON and HALL 1985). These plasmids were digested with BstEII prior to transformation in order to target integration to the genomic SUP4 locus. Plasmids used as templates for in vitro transcription reactions included pDA26-94 and pDA26-96 (ALLISON and HALL 1985) and PTC-U(IV) and PTC-SUP4. The latter two were derived from the vector pTC3 by the insertion of SUP4 alleles carried on 266-bp AluI fragments into a BamHI site (SHAW and OLSON 1984). The SUP#- AA36A37 allele was transcribed from a derivative of pBR322 with the 266-bp AluI fragment inserted into the BamHI site (ALLISON, GOH and HALL 1983). Retransformation of mutant candidates was done using pCU-U(IV), pCU-G37, and pCU-A94 (pCU indicates CEN, URA3), which are derived from the PTC and pDA26 series plasmids by insertion of the URA3 gene into the HindIII sites, replacing the T R P l gene in the process.

Genetic techniques: Yeast mating, sporulation, and tetrad analysis were carried out as described by SHERMAN, FINK and HICKS (1 983). Standard yeast transformation was by the method of BEGGS (1978); integrative transformation was according to ORR-WEAVER, SZOSTAK and ROTHSTEIN (198 1). In vivo suppression analysis was done by first patch- ing yeast onto YEPD plates, then replica plating after 1 day of growth to -lys and YEPD plates. Scoring of both lysine auxotrophy and ade2-1 color phenotype was done after incubation for 3 days at 30”.

Determination of integrated plasmid copy number: Yeast DNA was prepared from strains carrying integrated plasmids by the method of SHERMAN, FINK and HICKS (1983). Approximately 2 fig of DNA was digested by a restriction enzyme known to cut once within the plasmid sequence and electrophoresed on 1% agarose gels. The DNA was blotted by the method of SOUTHERN (1975) to a nitrocellulose membrane and was hybridized with nick- translated plasmid DNA, and an autoradiogram was developed. The presence of a plasmid-sized band is indicative of multiple plasmid integration. Two plasmid copies us. three plasmid copies was determined by comparison of band in- tensities within a lane; higher copy numbers could not be reliably distinguished. In vitro extract preparation and fractionation: I n vitro

transcription extracts were prepared as described by ALLI- SON, GOH and HALL (1983) with the following modifications. Yeast cells were broken with glass beads in the 30-ml chamber of a Bead Beater (Biospec Products) by one 20-sec burst. Although this yielded only -25% broken cells, longer breakage periods resulted in reduced extract activity. Dialysis of the extract was replaced by a desalting column of Sephadex G-25 packed to 5 ml and eluted with buffer C containing 100 mM KCI. Protein was monitored by Bradford assay (Bio- Rad) and peak fractions were pooled and stored at -70”. Elimination of dialysis also improved extract activity, espe- cially for strains which are not protease deficient.

Extracts which were to be fractionated were absorbed to a 5-ml phosphocellulose column equilibrated with buffer C plus 100 mM KC1 and step-eluted with buffer C plus 750

RNA Pol I11 Termination Mutant 295

mM KCI. Protein was assayed and peak fractions were pooled and desalted on a 5-ml Sephadex G-25 column with buffer c plus 100 mM KCl. Protein-containing fractions were loaded onto a 2-ml DEAE-Sephadex A-25 column equilibrated with buffer C plus 100 mM KC1 and step eluted with buffer C containing 300 mM and 1000 mM KCI. Peak protein fractions were pooled from the flow-through and each of the step elutions; the latter were desalted on Seph- adex G-25 columns with buffer C plus 100 mM KC1, and all were stored at -70". The flow-through fraction contained factor TFIIIB, the 300 mM fraction TFIIIC, and the 1000 mM fraction RNA polymerase 111. These were used for in vitro transcription reactions as described below.

RNA polymerase I11 purification: RNA polymerase 111 was purified by modification of published methods (VAL- ENZUELA et al. 1978; HUET et al. 1985). Cultures (100 liter) of yeast cells were grown in YEPD media to OD600 of 3.0 using a batch fermenter (New Brunswick Scientific) and harvested in a Sharples continuous flow centrifuge, yielding a 350-450 g (wet weight) cell pellet. All remaining steps were carried out at 0-4". All buffers used were solubuliza- tion buffer (200 mM Tris, pH 8.0; 10% glycerol; 10 mM MgCI2; 10 mM 8-mercaptoethanol; 1 mM phenylmethylsul- fonyl flouride) containing the indicated concentration of ammonium sulfate. The cell pellets were washed once in HpO and resuspended in 2 ml/g of 350 mM buffer. Cells were broken by five 30-sec bursts in the 350-ml chamber of a Bead Beater containing one-half volume of acid-washed 0.45-0.55-mm glass beads, with cooling in an ice-ethanol bath between bursts. This procedure resulted in -40% breakage, as judged by phase-contrast microscopy. The cell lysate was decanted and the beads were reextracted twice with an equal volume of 350 mM buffer. The combined lysate was spun at 10,000 rpm in a Sorvall GSA rotor for 10 min and the supernatant was recovered. Polymin P was added to a final concentration of 0.35% (w/v), stirred for 10 min, and spun in a GSA rotor at 10,000 rpm for 10 min to remove DNA. The supernatant was recovered and solid ammonium sulfate was added to 0.35 g/ml, stirred for 30 min and spun in a GSA rotor for 30 min at 10,000 rpm. The pellet was resuspended in 0 mM buffer and diluted until the salt concentration was 50 mM, as determined by conductivity. The extract was absorbed to a 400-ml DEAE- Sephadex A-25 column equilibrated with 50 mM buffer, washed, and eluted with a 3.5-column volume linear gradient from 50 to 500 mM. Column fractions were assayed for RNA polymerase activity as described; assays were done with and without 50 pg/ml a-amanitin and at 100 mM and 250 mM ammonium sulfate to distinguish between the ac- tivities of RNA polymerases I , I1 and 111 (ADMAN, SCHULTZ and HALL 1972). RNA polymerase I eluted in the flow- through while RNA polymerases I1 and 111 coeluted at 125 mM ammonium sulfate. Polymerase 111-containing fractions were loaded directly onto a 70 ml heparin-Sepharose column equilibrated with 250 mM buffer, washed, and eluted with a 3-column volume linear gradient from 250 to 750 mM. RNA polymerase I1 was eluted in the wash and RNA polymerase 111 at 500 mM. polymerase III-containing fractions were pooled and concentrated fivefold by centrifugation in Centriprep-30 microconcentrators (Amicon), then diluted with 0 mM buffer containing 10 mM EDTA to give a final salt concentration of 50 mM. The extract was loaded onto a 15 ml DNA-cellulose column (Pharmacia) equilibrated with 50 mM buffer plus 10 mM EDTA, washed, and eluted with a 5-column volume linear gradient from 50- 750 mM. RNA polymerase I11 activity eluted at 250 mM and was concentrated by centrifugation as just described and diluted with 0 mM buffer to 50 mM. The extract was rechromatographed on an 8-ml DEAE-Sephadex A-25 col-

umn using a 5-column volume linear gradient from 50 to 500 mM. Active fractions were concentrated 50-fold by centrifugation, adjusted to 70% glycerol, and stored at -70 " at concentrations of 1-1.5 mg/ml. Final yield was 300-400 pg of protein; purity was analyzed by gel electrophoresis according to the method of LAEMMLI (1 970) (see Figure 4). In vitro transcription reactions: Transcription reactions

were incubated at 25" for 30 min in a final volume of 20 pl. Each reaction included final concentrations of 20 mM HEPES-KOH, pH 7.9; 70 mM KCI; 5 mM MgC12; 0.5 mM dithiothreitol; 0.1 mM EDTA; 5% glycerol; 0.4 mM each of ATP, CTP and UTP; 0.05 mM GTP; 6pg/ml DNA template; and 12.5 p M [a-"P]GTP (400 Ci/mmol; Amersham). Standard reactions included 1-5 pl of crude in vitro transcription extract. Reconstituted transcription reactions contained 5 PI each of fractions containing TFIIIB and TFIIIC, and either 5 pl of a fraction containing RNA polymerase 111 or a final concentration of 1 pg/ml of purified RNA polymerase 111. All components were mixed on ice prior to the addition of transcription factors, and then transferred to 25". Recovery and electrophoresis of transcription products were carried out as previously described (BAKER and HALL 1984).

RESULTS

Selection of termination mutants: We have de- signed a selection system in yeast to isolate mutations which change the recognition specificity of transcription regulatory proteins. These mutations will lead to altered utilization of weak transcription termination signals and will be expressed phenotypically as an increase in the ochre suppression of auxotrophic markers. The selection procedure (Figure 1) employs a yeast strain carrying an ochre-suppressing allele of the SUP4 gene that has been transcriptionally inacti- vated by a cis-acting transcription terminator mutation. This inactive suppressor tRNA gene serves as a target gene to be reactivated by trans-acting mutations that raise or lower the termination efficiency of RNA polymerase 111. These mutations should identify genes encoding transcription termination proteins.

Though mutant selection was carried out in both haploid and diploid yeast, we concentrated our efforts on diploid selection for three reasons. First, we hy- pothesized that a mutant protein with altered signal recognition properties would behave as a gain-of- function mutant and be phenotypically dominant or codominant. Second, because most transcription proteins are likely to be essential, a mutation affecting specificity may be lethal in a haploid. Finally, approximately one-third of all ochre suppressing mutants that arise in haploid selections behave as allosuppressors (COX 1977); that is, they provide a nonspecific enhancement of all suppressor tRNA alleles. Because most allosuppressors are recessive, they will not be obtained in a diploid selection. In addition, we chose to select spontaneous events rather than to use mutagenic agents because of the wider range of single base substitutions produced (COULONDRE and MILLER 1977; KURJAN and HALL 1982; SCHAAPER, DANFORTH and GLICKMAN 1986).


-1ys 7 - 3odays

Plate popouts on -1ys

Saccharomyces cerevisiae strain PJ 14, defined in detail in Table 1, is homozygous for ura3-1 and the ochre suppressible markers met4-l,lys2-1 and ade2-1. These markers, in the order listed, require increasing levels of suppression for prototrophic growth (HAWTHORNE and LEUPOLD 1974). The degree to which each mutant isolated affects transcription termination can be judged by the auxotrophies it is able to suppress. In addition, development of red color due to the ade2-1 allele provides a continuous scale of suppressor strength. PJ14 is also heterozygous for the SUP4-G37 allele, an ochre suppressor which is defective but leaky due to a lesion affecting intron processing (COLBY, LEBOY and GUTHRIE 198 1). This allele was used to eliminate mutations that enhance a weak suppressor in ways not related to transcription termination.

Two different termination defective target alleles were introduced into PJ 14 cells and used for selection (see Figure 2 for diagrams). SUPI-U(ZV) contains a weak termination signal within its intron that causes the majority of the tRNA transcripts to terminate prematurely (KURJAN et a l . 1980; KOSKI et al. 1980). Decreased utilization of this termination signal is expected to produce more full length suppressor tRNA molecules, thereby increasing phenotypic suppression. The SUP4-A94 allele has a weakened terminator at the natural 3' end of the gene. In a wild-type background, many of the SUP4-A94 transcripts read through this termination signal to produce elongated pre-tRNA molecules; these are nonfunctional because their 3' processing is defective (ALLISON and HALL 1985). In the case of SUP4-A94 increased suppression

u -1ys

FIGURE 1 .-Mutantselection. PJ14 cells containing integrated YIp5- U(IV) (c,. YIp5-A94) plasmids were spread onto YEPD plates at a density of 200 cells/plate. Colonies were grown to -3 mm in diameter and were replica plated to either -lys or - ade media. The selective plates were incubated at 23". 30", or 36" for up to 30 days to allow spontaneous mutations to appear as papillations. N o more than one papillation from each colony was selected and patched onto complete synthetic media containing 5-flouro-orotic acid to select for excision of the YIp5-U(IV) integrated plasmid. Mutants for which loss of the SUP4 allele was accompanied by a loss of ochre suppression were retransformed with the same SUP4 allele used in step one of the selection. True trans-acting mutations in regulatory proteins are expected to regain ochre suppression, while cis-acting mutations to the original plasmid will remain auxotrophic after retransformation. Only one mutant was found to behave in a trans-acting, SUP4 allele-dependent manner.

will result from mutations which increase recognition of the weak terminator. Thus we are able to select for both increased and decreased utilization of weak terminators. Each of these SUP4 alleles is somewhat leaky and when present in single copy in a wild-type strain will suppress the met4-1 lesion of PJ14. In higher copy numbers these alleles will suppress 4x2-1 and ade2-1 as well. For this reason, absolute control of the target SUP4 allele copy number was necessary. The copy number control of CEN-based plasmids proved to be insufficiently stringent, leading to high background suppression. We integrated a single copy of the line- arized plasmid YIp5-U(IV) or YIp5-A94, carrying the URA3 marker gene and a SUP4 target allele, at the SUP4+ locus of diploid PJ14, a heterozygote containing the SUP4+ and SUP4-G37 alleles.

In total approximately 1 1,000 PJ 14 colonies (1 0" cells) were replica plated to selective media, and papillations which grew out from the colony "ghosts" were picked, yielding 7623 prototrophic mutants which could be restreaked on selective media. No more than one papillation from each colony was selected for analysis in order to avoid duplicate events. Most colonies gave one to three papillations, and new mutations which restreaked successfully continued to appear even thirty days after replica plating. There were 960 prototrophs obtained using the YIp5-A94 plasmid and 6663 with the YIp5-U(IV) plasmid. Of the latter group, 794 were selected in the haploid strain PJ6-3C carrying the same suppressible markers as PJ14; all others were selected in PJ14.

Each of the mutants was patched onto complete

RNA Pol 111 Termination Mutant 297

SUP4 - Q TABLE 1

Yeast strains

Strain Genotype

PJ6-3C

PJ1 1-12D

PJ14

PM3958

PJ17-IA

PJ 17-6A

PJ19

PJ19-33B

PJ2 1

PJ21-47D

PJ26

PJ26-6C

MATa SUP4-G37 trpl-I, ura3-1 ade2-I, lys2-I, met4-I, canl-100.leul-12

MATa trpl-I, ura3-1 ade2-1, lys2-1, met4-I, canl- 100.leul-12

MATalMATa SUP4-G37/+ trpl-I, /+ trp5-20/+ ura3-l/ura3-l ade2-I0/ade2-l . lys2-I, / lys2-I0 met4-lO/met4-l. canl-100./canl-100, leul-121 leul-12

MATa [URA3::SUP4-U(IV)]. ret l -1 t r p l - I . ura3-1 ade2-I, lys2-I, met4-I, canl-100, led-12

MATa trpl-I. ura3-1 ade2-1, lys2-1, met4-I, canl- 10O0gall0- l , his5-2: leu2-I1

MATa trpl-I . ura3-1 ade2-1 1 s2-1, met4-I. canl- 100 ,ga l l0 - lU his5-21 leu2-1, , y

MATaIMATol [URA3::SUP4-lJ(lV)],/+ re t l - I /+ trp1-1Jtrp1-Ia ura3-1/ura3-1 ade2-l,/ade2-l0 lys2-1,/lys2-1, met4-I./met4-l0 canI-I00./canI- 100, leul-12/ leul-12

MATa retl-1 trpl-1, ura3-1 ade2-I, lys2-1. met4-I, canl-100. leul-12

MATalMATa r e t l - I / + t r p l - l a / t r p l - l a u r a 3 - l / ura3-1 ade2-l0/ade2-l, lys2-l0/lys2-I, met4-l,/ met4-1. canI-100,/canI-100. leul-12/+ leu2-11/ + his5-21/+ gallO-I,/+

MATa retl-1 trpl-I, ura3-1 ade2-I, lys2-I, met4-I, canl-100, his5-21 leu2-1:

MATaIMATol re t l - I /+ t r p l - l a / t r p l - l ~ u r a 3 - I / ura3-1 ade2-1,/ade2-I0 lys2-I,/lys2-I0 met4-I./ met4-1, canl-100./canI-10O0 leuI-12/leuI-12

MATa retl-1 t rp l - I , ura3-1 ade2-I, lys2-1, met4-I, can l -100 , l eu l -12

synthetic media containing 5-flouro-orotic acid to select for excision of the URA3-SUP4 integrated plasmid (BOEKE, LACROUTE and FINK 1984). In 22% of the mutants, loss of the SUP4 allele was accompanied by a loss of ochre suppression. The remaining 78% showed prototrophic growth independent of the plasmid, and are expected to include mutations such as new ochre suppressors, revertants of SUP4-G37, and revertants of auxotrophic markers. Mutants depen- dant on the SUP4 termination-defective allele were retransformed with the same SUP4 allele used in the initial selection carried on a CEN plasmid (pCU-U(1V) or pCU-A94), which proved suitable for this step, and also with pCU-G37 to distinguish suppression effects that were not allele specific. True trans-acting mutations in regulatory proteins would be expected to regain ochre suppression with pCU-U(1V) or pCU- A94, but not with pCU-G37. Clones originating from plasmid-borne, cis-acting mutations should remain auxotrophic with both SUP4 alleles. Over 300 mutants, or about 20% of all selected, were retransformed before finding one which regained suppression; the rest appeared to be cis-acting mutations in the plasmid-born target allele. No true mutants were identified from the haploid selection. Only PM3958, which arose from the diploid PJ14 containing the

SUP4 - 3’ A ’S \ \ L T 7 G T S

FIGURE 2.”SUP4 termination-defective alleles. For each of the four termination-defective alleles used in this study, arrows indicate the nucleotides affected; the size of the resulting T tract is indicated in parentheses below the allele name. The general structure of the SUP4-3‘A alleles was previously described (ALLISON and HALL 1985). The A box and B box denote the positions of the internal promoter elements, AC indicates the anticodon, and parentheses show the position of the intron.

YIp5-U(IV) plasmid, was found to behave in a trans- acting, SUP4 allele-dependent manner. T o confirm this phenotype, an SlOO extract from this mutant was tested in an in vitro transcription assay, as described below, and was observed to have altered transcription termination.

At least three genetic events are responsible for ochre suppression in PM3958: As the single mutant clone that survived the selection procedure, PM3958 originated from a PJ14 diploid containing the YIp5- U(1V) plasmid, which was incubated on a -1ys plate for 21 days at 30”. We have determined that PM3958 is the product of at least three rare events, including the mutation itself, an uninduced sporulation, and amplification and recombination events which may or may not be separate. We have also determined that each of these events was necessary for expression of the suppression phenotype and for the recovery of the mutant.

Though it arose from a diploid selection, PM3958 is in fact a haploid. It contains all of the homozygous markers and two of the three heterozygous markers of PJ14, as well as the a-mating type, suggesting it is a normal meiotic segregant of PJ 14. We used mating tests to check the ploidy of the parent strain and of several dozen other mutant isolates originating on the same selective plate as PM3958, and found all to be diploid. As described below, suppression in PM3958 is dependent upon a single mutation which is unlinked to the SUP4 locus. We have crossed the PM3958 mutation into diploid backgrounds containing as many as five copies of the SUP4-U(IV) allele and have not observed any differences in ochre suppression between heterozygous and homozygous wild-type cells, using both the lys2-I and the ade2-1 alleles. Therefore the PM3958 mutation is recessive to wild


1 2 3 4 5 6 7 8 9 1 0 I

Readthrough - Transcript

Gene Length - Transcript

type, and haploidization was absolutely required for ochre suppression to be expressed in PM3958.

In addition to mutation and haploidization, there is evidence for the creation of multiple tandem repeats at the SUP4 locus, making the SUP4 locus of PM3958 different from those of either homolog of PJ14. Hy- bridization analysis (SOUTHERN 1975) using probes from the SUP4 region allowed us to demonstrate that PM3958 has undergone an amplification of the entire integrated YIp5-U(IV) plasmid, resulting in a mini- mum copy number of three (data not shown). As was the case with haploidization, the parent PJ14 and several mutant isolates originating on the same plate as PM3958 were checked by Southern hybridization analysis and all contained single copies of the integrated plasmid. Further information about the event is provided by a restriction site polymorphism in the SUP4 region of the two chromosome 10 homologs of PJ14. PM3958 was shown to carry the polymorphism previously linked to the chromosome 10 homolog of PJ14 carrying the SUP4-G37 allele, indicating that a recombination event had occurred between the homologs of PJ14 prior to haploidization. We cannot determine whether this recombination and the amplification of the integrated plasmid occurred as a single event .

In subsequent crosses to strains containing multiple integrated YIp5-U(IV) plasmids we have determined that the optimal copy number for observation of the suppression phenotype conferred by the mutation is two. One copy of YIp5-U(IV) does not allow any suppression in the presence of the mutation; thus,

- Transcnpt Gene Length

- Premature

- Products Termination

FIGURE .?.-In vitro transcription of SUP4 ter- Inination-defective ;~lleles. SUP4 template genes were transcribed in vitro using wild-type (odd-numbered lanes) and ret l -1 (even-numbered lanes) S I00 extracts and electrophoresed on a 7 M urea, 10% polvacr~l;~micle gel. l a w s 1 and 2. SUP4-AY6: lanes 9 and 4, SUP4-AY4: lanes 5 and 6. SUP4-0: lanes 7and X. SUP4-U(IV): lanes 9 and 10. StiP4- U 3 6 A 3 7 .

amplification also was required for the suppression phenotype of PM3958 to be expressed. In fact, PM3958 might not have been detected had retransformation been carried out with integrating rather than CEN-based plasmids with their leaky copy number control. Three or more copies of YIp5-U(IV) produce background suppression even in wild-type strains, although the ade2-1 color phenotype can still be distinguished. Finally, the PM3958 mutation was crossed into the diploid PJ21, which is homozygous for the lys2-I marker and heterozygous for the PM3958 lesion. A PJ21 diploid containing two copies of the YIp5-U(IV) plasmid was sporulated and 101 tetrads were analyzed. Tetrads were scored as being 0:4, 1:3, or 2:2 for growth:no growth on -lys media. These classes occurred in the ratio 1 :4: 1, as expected for a phenotype dependent on the segregation of two independent Mendelian loci (ie., SUP4 and the PM3958 mutation).

We conclude that the PM3958 mutant clone was recovered as the result of at least three separate, rare events: the mutational change itself, an uninduced sporulation, and amplification of the suppressor tRNA gene. All three of these events are essential to the expression of the suppression phenotype and thus to the detection of the mutation. A recombination event at the SUP4 locus may or may not have been concurrent with the amplification event. In vitro phenotype of retl-I: In vitro transcription

assays were carried out using S 100 extracts made from wild type and PM3958 cells and five different tRNA templates. Transcription assays and extracts are as


described in MATERIALS AND METHODS. The five tern- plates transcribed include the transcriptionally wild- type SUP4-o gene and four termination-defective alleles, depicted in Figure 2. The mutations in two of the alleles, SUP4-U(ZV) and SUP4-DA36A37, create new termination signals internal to the gene that cause premature termination and shortened transcripts (KO- SKI et al. 1980). The SUP4-A94 and SUP4-A96 alleles contain shortened T tracts at the normal terminator location, causing read-through and elongated, unprocessed transcripts (ALLISON and HALL 1985).

Figure 3 displays the transcription patterns of each of these alleles in extracts from both wild-type and PM3958 cells. Differences in total transcription activity between wild-type and mutant extracts appear to be due to variation in individual extracts. Each template except SUP4-o yields two major bands corre- sponding to a longer and a shorter transcript. In each case, transcription by the PM3958 mutant extract produces an increased percentage of total signal ap- pearing in the larger transcript class, as compared with the adjacent wild-type lane. Thus, the amount of read-through transcription of the SUP4-A94 and SUP4-A96 templates is increased in extracts made from PM3958 cells, as compared to extracts made from wild-type cells (compare lanes 1 and 2, 3 and 4). The reduced use of weak terminators in mutant extracts is clearly seen for the SUP4-Cr(ZV) allele with which the mutant was isolated. Premature termination products from this template observed in wild-type extracts (lane 7) appear at a much lower frequency in extracts from strain PM3958 (lane 8). The same effect is seen with the SUP4-AA36A37 template (lanes 9 and 10). This demonstrates that in PM3958 utilization of weak termination signals is reduced, resulting in increased read-through to more distant termination signals. Accordingly, we have named the gene that is mutated in PM3958 RETl, for reduced efficiency of termination, and the mutant allele retl-1. Separate experiments have shown that in vitro read-through at the SUP4-o terminator is <0.5% in both retl-I and wild-type extracts (S. SHAABAN, personal communi- cation). In vitro termination efficiency was quantified by densitometry and is presented in Table 2. All figures are adjusted for the number of labeled GTP residues which are incorporated into each transcript class. In vivo phenotype of retl-1: as described earlier,

tetrad analysis was used to demonstrate that retl-1 behaves as a single Mendelian locus. We tested retl-1 strains for a variety of growth defects. Neither sporulation or mating efficiency is affected by the retl-1 mutation. No ts or cs phenotype was detectable at any temperature at which related wild-type strains would grow. In addition, the doubling time of mutant strains was indistinguishable from wild type. Note that be-

TABLE 2

retl-1 termination phenotypes

Genotype Percent in vitro transcripts

SUP4 Gene Read- In vivo lys2-1

RETl Premature length through suppression

SUP4-0 RET1 0 100 0 +++ retl-1 0 100 0 +++

SUP4-U(IV) RET1 61 39 0 - retl-1 15 85 0 +

SUP4-AA36A37 RETl 87 13 0 NA"

ret l -1 40 60 0 NA

SUP4-A94 RETl 0 30 70 + re t l - l 0 14 86 -

SUP4-A96 RETl 0 91 9 ++ r e t l - I 0 65 35 ++

a NA = not applicable.

cause of the way this mutant arose, no isogenic parent strain exists for comparison.

Along with the in vitro data, Table 2 displays the in vivo phenotype of the retl-1 mutation in combination with each SUP4 allele except SUP4-AA36A37, in which the anticodon has been destroyed. As predicted, the termination phenotype of retl-1 can affect in vivo suppression in two ways in response to different SUP4 alleles. In a SUP4-U(ZV) background, the retl-1 mutant causes increased read-through, increased amounts of gene-length transcript, and thus a LYS+ phenotype in vivo. However, in a SUP4-A94 background, the increased read-through caused by retl-1 results in more unprocessed read-through transcript and less gene- length transcript, causing lysine auxotrophy. The data in Table 2 seems to present a paradox, as 30% gene- length transcript in a RETl, SUP4-A94 background allows suppression of the lysine marker, while 39% gene-length transcript in a RETl, SUP4-U(ZV) background is auxotrophic. However, it must be noted that gene-length transcript levels presented here may not accurately reflect in vivo levels. In vitro termination frequency is dependent on reaction conditions which include nucleotide, magnesium, and salt concentrations as well as temperature (BOGENHAGEN and BROWN 198 1 ; our unpublished results). Furthermore, mutations at many sites in a tRNA gene can alter the level of functional transcript accumulation by affecting processes such as transcription initiation, transcript stability and processing, or aminoacyl-tRNA synthetase recognition (YARUS 1988; KURJAN et al. 1980; ALLISON and HALL 1985; GIROUX et al. 1988). However, as was the case with SUP4-U(ZV), at least two copies of the SUP4-A94 allele are required for ochre suppression in a RETl strain in vivo. Thus, it appears that a doubling of the in vivo levels from these alleles is sufficient to cause suppression of Zys2-1.


retl-l is a mutant in RNA polymerase 111: In order to determine what part of the transcription complex is responsible for the retl-1 phenotype, we used fractionated transcription extracts from retl-1 mutant and wild-type yeast cells to do mixing experiments in a reconstituted in vitro transcription system. Extracts were loaded onto DEAE-Sephadex columns and step eluted to obtain retl-1 and wild-type crude fractions containing factors TFIIIB, TFIIIC, and RNA polymerase I11 (KLEKAMP and WEIL 1982). Transcription tests were done to verify that no single or double combination of fractions was active, but that reconstitution of all three fractions led to specific transcription. Complete reconstituted transcription systems were then made by mixing retl-1 and wild-type fractions in all combinations and transcribing the SUP4- U(IV) template. In each reconstitution that included the wild-type polymerase 111 fraction, normal transcription termination was observed, while those using retl-1 polymerase all exhibited increased read- through (data not shown; see Figure 5 below). There was no correlation between the termination phenotype and the source of either TFIIIB or TFIIIC. These results demonstrate that the retl-1 in vitro termination phenotype is dependent on the source of the RNA polymerase 111 fraction, and independent of the TFIIIB and TFIIIC fractions.

The reconstitution experiments strongly suggest that RNA polymerase I11 is altered by the retl-1 mutation, since read-through transcription is in all cases correlated with the presence of the retl-1 polymerase. However, in these experiments we used crude protein fractions which might contain some previously unresolved RNA polymerase 111 transcription factor. T o address this possibility, we repeated the reconstitution experiment using highly purified RNA polymerase 111 preparations from retl-1 and wild-type yeast. An sodium dodecyl sulfate (SDS)-polyacrylamide gel of the polymerase preparations is shown in Figure 4; arrows indicate RNA polymerase 111 subunits whose genes have been cloned. The RNA polymerase 111 enzyme is made up of 14 to 15 different protein subunits, all except one of which are present in a single copy in the holoenzyme (VALENZUELA et aZ. 1976; SENTENAC 1985). No obvious differences such as missing or truncated proteins are visible between the retl-1 and wild-type enzymes. The purified polymerases were used in a reconstituted transcription system with the crude fractions containing TFIIIB and TFIIIC and with SUP4-U(IV) as template. The results are shown in Figure 5. As was observed with the crude polymerase fractions, the termination phenotype is determined by the source of RNA polymerase 111. Therefore the RET1 locus is likely to encode one of the subunits of RNA polymerase 111, although these results do not rule out the possibility that it encodes

1 2

FIGURE 4.-Purified RNA polymerase 111. RNA polymerase 111 was purified a s described in MATERIALS AND METHODS from wild- type (lane 1) and retl-1 (lane 2) cells. A 21-pg sample of each polymerase w a s run on a split SDS-PAGE gel of 15% (lower 2/3) and 12% (upper 1/3) polyacrylamide and stained with Coomassie blue. Arrows indicate polynlerase subunits which have been previously cloned.

an enzyme that covalently modifies a core subunit of the polymerase.

DISCUSSION

We have developed a selection system which requires a mutational change in a transcription regulatory protein to compensate for a defective DNA regulatory sequence. We have used this system to inves- tigate transcription termination; however, it should be possible to modify the selection to any situation in which the interaction of a protein with a regulatory site can be linked to a selectable phenotype. Using this system, we have identified the recessive mutation retl-1 which changes the efficiency with which RNA polymerase 111 can terminate transcription.

The original mutant isolate, PM3958, was detected as the result of a series of rare events which, in combination, suppress the ochre mutation Zys2-I. It is clear that each of these events was necessary for the suppression phenotype to appear. We cannot determine the order in which these events occurred in the PM3958 isolate, however certain observations may provide clues. PM3958 is a haploid in which the YIp5- U(IV) plasmid was amplified to a copy number of at least three. In the presence of three copies of the YIp5-U(IV) plasmid, a haploid will grow weakly on -lys media even in a RET1 background, forming small colonies in about 5 days. However, PM3958 did not appear until 16-2 1 days of growth. Thus it is unlikely that the retl-1 mutation could have been the final event, since a cell in which both haploidization and

RNA Pol I 1 1 Termination Mutant 30 1

. 1 2 3 4 5

Gene Length - Transcnpt

Premature Terminatlon -

Product

FIGURE .i.-In vitro transcription reconstituted from fractionated TFIIIR;1nd TFllIC and purified R N A polymerase 111. T h e S U P 4 U ( l V ) allele w a s transcribed in vitro using the following: lane 1 , RETI SI00 extract; lane 2, crude fractionated TFl l lB and TFl l lC and purified RET1 RNA polymerase 1 1 1 ; lane 3, crude fractionated TFIIIB and TFIIIC, no polyn~erase; lane 4, crude fractionated TFIl lB and TFll lC and purified retl-I RNA polymerase I l l ; lane 5, retl-I SI00 extract. All T F l l l B and TFIl lC \cas isolated from retl-1 cells.

amplification had already occurred would have formed a colony in the absence of the mutation.

We reconstructed several possible intermediates in the creation of PM3958 to try to determine how these events might have occurred. When diploid yeast heterozygous for both retl-1 and SUP4-U(IV) are plated on -lys media approximately 800 prototrophs appear per lo7 cells, and 70% of these are haploid. However heterozygous diploids containing either retl-1 or SUP4-U(ZV), but not both, yield only 10-50 prototrophs per 10' cells, less than 1% of which have become haploid. T h e high frequency of prototrophy in the former case is due to the selective advantage of the meiotic products, since sporulation frequency is not affected by the retl-1 mutation (see RESULTS). Thus, given an appropriate background, sporulation on noninducing media is a high frequency event leading to suppression. Additionally, the recombination event observed at the SUP4 locus, which may be associated with the amplification, certainly must have occurred while a homolog was still present. These observations suggest that the haploidization occurred concurrent with or later than the other events. One

possibility is that the recombination, amplification, and haploidization all occurred during a single meiosis, and that only the mutation itself preceded this.

T h e retl-1 mutation causes a phenotype of reduced termination efficiency at weak termination signals both in vivo and in vitro. In vitro, the mutant effect is more pronounced when weak terminators are used than for strong termination signals. When the template has the SUP4 wild-type termination signal, retl- I has essentially no effect. In vivo, retl-1 has no effect on the doubling time, temperature sensitivity, sporulation, or mating efficiency of yeast. By comparison with RETI strains carrying various copy numbers of SUP4 alleles, we are able to estimate that the in vivo effect of retl-1 on ochre suppression is approximately twofold. The apparent weakness of the retl-1 phenotype is a logical and necessary outcome, given the way this selection was done. The mutation was required to affect weak terminators, but a mutation that also affected utilization of the strong termination signals present in most normal genes would have caused gross alterations in tRNA and 5s gene expression levels and could therefore have been lethal. This may also ex- plain why mutations were not recovered at a higher rate in this selection, since most would have effects either too weak to produce a suppression phenotype or too strong to be viable.

Fractionation and reconstitution of in vitro transcription extracts has demonstrated that the retl-1 phenotype is mediated by RNA polymerase 111 itself. This result supports the conclusion of COZZARELLI et al. and of WATSON, CHANDLER and GRALLA that the polymerase is directly involved in the recognition of the termination signal. I t does not imply that the polymerase alone is sufficient for termination, nor does it rule out the possibility of a yeast analog to the mammalian La protein. It should be noted that efforts to identify a yeast protein with immunological similar- ity to La have been unsuccessful, but a poly-U binding activity has been observed (D. BROW, personal com- munication).

One model that would reconcile our results with those of GOTTLIER and STEITZ (1989b) supposes that RNA polymerase 111 is necessary for the signal recognition and pausing step(s) of transcription termination, and that La or a La-like protein is necessary for the release step. Alternatively, the yeast RNA polymerase 111 system may carry out termination without any protein factor equivalent to La, in which case yeast RNA polymerase 111 is both necessary and sufficient for transcription termination. Such a system might be the reason that yeast requires a more extensive T tract to signal efficient termination than do higher eukaryotes.

Structural similarities have been demonstrated between the subunits of a number of RNA polymerase


enzymes including RNA polymerase 111, and it is expected that these subunits will show functional sim- ilarity as well (HUET, SENTENAC and FROMAGEOT 1982; ALLISON et al. 1985; SWEETSER, NONET and YOUNG 1987). There are several functions in the transcription process that retl-1 might affect in order to produce the observed termination phenotype. For example, altered binding to the DNA template, the nascent RNA, or the nucleotide substrates, or changes in the elongation rate or in termination signal recognition might affect termination rates. In Escherichia coli, two proteins associated with the RNA polymerase have been implicated in transcription termination. a collection of rifampicin-resistant mutants in the 0- subunit of E. coli RNA polymerase have been shown to affect termination by that enzyme (JIN and GROSS 1988; JIN, WALTER and GROSS 1988); the authors suggest that the primary effect of the rifR mutants may involve nascent RNA or nucleotide binding. Also, the NusA protein, which is associated with the core polymerase during elongation, has been shown to enhance pausing by RNA polymerase (for review see PLATT 1986).

While our in vitro reconstitution experiments have shown that the retl-1 termination defect is associated with the RNA polymerase enzyme, rather than with a dissociable protein factor, any one of the protein subunits seen in Figure 4 might be affected. Four of these (indicated by arrows in Figure 4) have previously been isolated in molecular cloning experiments (RIVA et al. 1986; MANN et al. 1987; GUDENUS et al. 1988). We used transformation of retl-1 yeast strains by plasmids bearing the wild-type alleles of these RNA polymerase I11 subunits to determine which of them, if any, encodes the RETl gene. Transformants containing the C160, C53, AC40 and C3 1 clones all remained retl-1 in phenotype, suggesting that the retl-1 mutation affects another, previously uncloned subunit of RNA polymerase 111.

The retl-1 mutation is the first identified which affects eukaryotic transcription termination, and it demonstrates the importance of the RNA polymerase enzyme itself for in vivo termination. Further analysis should lead to an enhanced understanding of both the polymerase enzyme and the process of transcription termination. Using the unique ability of the RETl gene to cause both increased and decreased ochre suppression, we have isolated a clone from a yeast genomic library (ROSE et al. 1987) which is able to complement the retl-1 mutation in vivo. The molecular characterization of that clone will be described in a forthcoming paper.

We wish to thank PIERRE THURIAUX, ANDRE SENTENAC and CARL MANN for providing clones of polymerase subunits. We are very grateful to CARL MANN and RICHARD BAKER for many helpful discussions regarding this work, and to MALCOLM WHITEWAY for advice concerning allosuppressors. We also thank ELIZABETH GRAVES-FURTER for careful reading of the manuscript.

This work was supported by research grant GM 1 1895 from the National Institutes of Health (GM62-0379). P.J. was supported by training grant GM07735 from the National Institutes of Health.

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retl-1, a yeast mutant affecting transcription termination

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