Conditional Mutants of RPCl60, the Gene Encoding the ... · transformation of the E. coli strain BW310, about 2000 pooled ampicillin-resistant transformants were harvested in LB supplemented

Copyright 0 1988 by the Genetics Society of America

Conditional Mutants of RPCl60, the Gene Encoding the Largest Subunit of RNA Polymerase C in Saccharomyces cerevisiae

Rosmarie Gudenus,' Sylvie Mariotte, Alejandra Moenne, Anny Ruet, Sylvie Memet, Jean-Marie Buhler, Andre Sentenac and Pierre Thuriaux

Service de Biochimie, Dbpartement de Biologie, C.E.N. de Saclay, Commissariat a I'Energie Atomique, F91191 G$ sur Yvette (cedex), France

Manuscript received September 24, 1987 Revised copy accepted March 12, 1988

ABSTRACT A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA

polymerase C (RPCI6O) was cloned by hybridization to a previously isolated fragment of that gene. RPG160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSOs and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpcl60 mutants by failure to complement a fully defective mutation rpcl60:: URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. I n vivo, the rpcl60 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5s rRNA and no effect on 5.8s rRNA. An unlinked mutation (pci l -3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

I N yeast as in all other eukaryotes, the genes of small RNAs such as tRNA and 5s rRNA are

specifically transcribed by one of the three nuclear RNA polymerases, RNA polymerase C (or 111). This enzyme has been extensively purified and contains fourteen distinct polypeptides (hereafter called subunits), of which some are shared with RNA polymerase A or with both RNA polymerase A and B (SEN- TENAC 1985). The promoters recognized by RNA polymerase C are intragenic. For transfer RNAs corresponding to nonsense suppressors, mutations with defective promoters lead to an antisuppressor phenotype and have therefore been relatively easily isolated and characterized in the yeasts Saccharomyces cerevisiae (ALLISON et al. 1983) and SchizosaccharomyceJ pombe (PEARSON et al. 1985). The intragenic promoters are not directly recognized by RNA polymerase C itself, but by additional proteins (transcription factors) which must be added to the purified RNA polymerase C to achieve faithful transcription in vitro (KLEKAMP and WEIL 1982; KOSKI et al. 1982; RUET et al. 1984; CAMIER et al. 1985) and are therefore essential to the gene specificity of RNA polymerase C.

In spite of extensive enzymological and immunological studies (SENTENAC 1985), little is known about the interaction between RNA polymerase C and its cognate transcription factors. Moreover, it is not clear

Donau, Austria. ' Present address: Immuno AG, Uferstrasse 15, A2304, Orth an der

whether the yeast RNA polymerase C participates in the transcription of genes other than those corresponding to tRNA and 5s rRNA. Given the impor- tance of yeasts as model systems to study major cellular processes in eukaryotes, we have undertaken the isolation and characterization of mutants pro- ducing a conditionally defective RNA polymerase C in S. cerevisiae. Ten genes potentially corresponding to distinct subunits of RNA polymerase C have been cloned by immunological screening (RIVA et al. 1986). One of them (RPC160) encodes the largest subunit C160 and is identical to a gene previously identified by its homology to the structural gene of the largest subunit of RNA polymerase B in Drosophila melano- gaster (INGLES et al. 1984). [RNA polymerase subunits are symbolized by the capital A, B or C followed by a number corresponding to their estimated molecular weight x 10'. Several symbols have been used to designate the corresponding genes (RUET et al. 1980; INCLES et al. 1984; RIVA et al. 1986; NONET et al. 1987). Pending a general agreement on this, we and others (MANN et al. 1987) have adopted the nomenclature proposed by RIVA et al. (1986).] Both genes have a striking sequence homology with the gene encoding the largest subunit of bacterial RNA polymerase (ALLISON et al. 1985). We report here the construction of temperature-sensitive mutant alleles of RPCl60 that affect the synthesis of tRNAs in vivo and in vitro, the isolation of an extragenic suppressor restoring growth at the restrictive temperature and the precise localization of the RPC160 gene on the chromosome

Genetics 119 517-326 (July, 1988).

518 R. Gudenus et al.

TABLE 1

Yeast strains

Name Genotype

YNN281"

YNN281canRb

YNN282"

YNN281-15

YNN281-20

YNN281-31

YNN281-41

D27-7C

YNN281-41-3

D25-13A 382-23A' 393-35C' 381-9D"

396-22B'

Be3 1 4d NC93SPl' T12-2B(pSP4)

20B-12f

CMY214' D8 D9 Dl0 T2 TDU TD9 TDlO

D25-13A

ura3-52 his3-200 trpl-901 lys2-801 ade2-101



uru3-52 his3-200 trpl-901 lys2-801 ade2-I01

ura3-52 his-3-200 trpl-901 lys2-801 ade2-101


ura3-52 his3-200 trpl-901 lys2-801 ade2-I01

ura3-52 his3-200 trpl-901 lys2-801 ade2-I01

urd-52 his3-200 trpl-901 lys2-801 ade2-I01

his3-200 trpl ade2-I01 pep4-3 rpc160-31 MATa spoll ura3-52 can1 cyh2 ade2 hi57 h o d MATa spoll ura3-52 his2 leu1 lysl met4 pet8 MAT spol1 ura3-52 ade6 arg4 a707 asp5 met14 lys2

spoll ura3-52 adel his1 leu2 lys7 met3 trp5

tsm 8740 MATa ura3-351,273,328 MATa ura3-52 his3-200 trip1 -901 lys2-801 ade2-IO1

rpcl60 ; : URA3+ MAT (pSP4) pep4-3 trpl MAT pep4-3 trpl his3-200 ade2-I01 MAT YNN281 canR X YNN282

MATa

canR MAT

MAT

rpc160-15 MATa

rpcl60-20 MATa

rpcl60-31 MATa

rpcl60-41 MATa

rpc160-41 MATa pep4 :: HIS3+ MAT

rpcI60-41 MATa pcil-3 MATa

pet1 7 trpl MAT

MAT

382-23A X 393-35C 382-23A X 396-22B 382-23A X 381-9D rpcl60 :: URA3 + transformants of CMY2 14 rpcl60:: URA3 + transformants DU rpcl60 : : URA3 + transformants D9 rpcl60 : : URA3 + transformants D 10

Yeast Genetic Center, Berkeley.

KLAPHOLZ and EASTON-ESPOSITO (1982). HILGER, PREVOT and MORTIMER (1982). F. LACROUTE, unpublished data.

' MANN et al. (1987).

JONES (1976).

XV. In a parallel work, mutations of the RPC40 gene (which encodes a subunit shared by RNA polymerase A and C) have been obtained, and were shown to affect the in vivo assembly of both RNA polymerases (MANN et al. 1987). Temperature-sensitive mutants of the gene encoding the largest subunit of yeast RNA polymerase B have also been described recently (HIMMELFARB, SIMPSON and FRIESEN 1987; NONET et al. 1987).

MATERIALS AND METHODS

Yeast strains: The yeast strains used in this study are listed in Table 1. The standard genetic techniques for yeast

were described by SHERMAN, FINK and HICKS (1979). The proteinase-less phenotype of the pep4-3 mutation was followed by the method Of JOHNSTON (1977) using 2% com- mercial powder milk as caseine source. Strain D27-7C carries a pep4::HIS3 + null allele (kindly sent by C. WOOL- FORD) instead of pep4-3. Free spores from canRICAN+ heterozygous diploids were obtained by digesting the asci with 100-fold diluted Helix pomatia juice (Industrie Biolo- gique Francaise) for at least 1 hr at 30°, checking for complete digestion under the microscope and separating the aggregated spores by sonication. The spores were plated on a selective medium containing canavanine to isolate canR haploid segregants (KLAPHOLZ and EASTON-ESPOSITO 1982), with occasional formation of MATclIMATa diploids due to the mating of adjacent spores upon plating. Yeast transformation was done according to ITO et al. (1983). Integrative transformants are symbolized by :: before the symbol of the integrated DNA fragment (e.g., rpcl60:: URA3 + ). Transformants were distinguished from potential revertants or suppressors (such as obtained when transforming the amber lys2-801 mutants) by testing the mitotic instability of the transformed character and, if needed, by genomic hybridization analysis with an appropriate "P-labeled probe.

Plasmids and phages: Phage and plasmids A EMBL3 (FRISCHAUF et al. 1983), pSP65 (MELTON et al. 1984), YRp7 (PARENT, FENIMORE and BOSTIAN 1985) and YCp631 (BARNES and THORNER 1986) have been previously described. pEMBLYi32 is similar to pEMBLYi21 but contains the pUC19 polylinker (BALDARI and CESARENI 1985 and personal communication). Plasmid p02 is a pPBR322 de- rivate in which the 1881 bp EcoRV PvuII fragment has been deleted and a tandem du lication of a 635-bp ClaI fragment carrying the tRNA$ yeast gene [originating from plasmid pY20 (FELDMAN, OLAH and FIEDENREICH 1982)] has been inserted at the ClaI site (0. GABRIELSEN and C. MARCK, personal communication). Several plasmids carrying inserts overlapping with RPC160 were constructed in this study (Figure 1). pSPl is derived from pSP65 and carries the 2.5-kb EcoRI insert of RPC160 interrupted by a 1. l-kb Hind111 URA3 + fragment which was inserted by blunt-end ligation into the StuI site. YIp32-1 derives from pEMBLYi32 by the insertion of a 5.3-kb XbaI fragment which starts 1.2 kb upstream the initiation codon ofRPCI60 and ends within the coding sequence. Finally, pSP4 carries a 14.4-kb fragment containing the whole RPC160 gene inserted at the Sal1 site of YCp631. This fragment was isolated from a genomic library (M. SNYDER, unpublished data) of the yeast strain X2180C which contains Sau3A partial fragments cloned between the BamHI site of A EMBL3 (this library was obtained from C. MANN). Recom- binant phage and plasmids were obtained by standard techniques (MANIATIS, FRITSCH and SAMBROOK 1982). Plas- mids were kept and amplified in the Escherichia coli strains RR1 and HB 101 (MANIATIS, FRITSCH and SAMBROOK 1982). For NaHSOs mutagenesis, we used the E. coZi strain JM107 (YANISCH-PERRON, VIEIRA and MESSING 1985) to prepare single stranded DNA and the ung strain BW310 [defective in uracil N-glucosylase (DUNCAN, ROCKSTROH and WARNER 1978)] for the amplification of the mutagenized DNA.

Media and growth conditions for S. cerevisiae: Yeast strains were grown at 30" using standard media and growth conditions (SHERMAN, FINK and HICKS 1979). Temperature sensitive mutants were grown on YPD at 25" and tested at 37". Nutritional requirements were determined on minimal medium SD supplemented with the appropriate nutrients (SHERMAN, FINK and HICKS 1979). Except for tryptophan, the amino acid requirements are also met by 0.5% casamino

RNA Polymerase Mutants in Yeast 5 19

acid. Respiratory deficient mutants, resistance to canavanine and to cycloheximide were tested as described by KLAPHOLZ and EASTON-ESPOSITO (1982). Colonies resistant to 5-fluoroorotate were selected as described by BOEKE, LACROUTE and FINK (1985) using 50-mm Petri dishes incubated 5 days at 25". Sporulation was done on solid medium with K-acetate (10 g/liter) supplemented with the appropriate nutrients at one quarter of the concentration used for the SD medium.

NaHSOS mutagenesis: Single-stranded DNA was prepared from a 100-ml culture of JMlOl (YIp32-1) super- infected with phage IRl as described by DENTE, CESARENI and CORTESE (1983). 10 pg DNA were treated 10 min at 37" with 3.0 M NaHSO3 (SHORTLE and BOTSTEIN 1983) and the mutagen was removed by spun-column chromatogra- phy on a 1-ml Sephadex G-50 medium column. Upon transformation of the E. coli strain BW310, about 2000 pooled ampicillin-resistant transformants were harvested in LB supplemented with ampicillin and double-stranded plasmid DNA was prepared by alkaline lysis and CsCl density gradient (MANIATIS, FRITSCH and SAMBROOK 1982). The uru3-52 yeast strain YNN281 was transformed by about 10 pg of mutagenized plasmid DNA linearized at the unique CZaI site to promote genomic integration by targeted crossing-over at the R P C l 6 0 gene (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981). About 5000 transformants selected on uracil omission medium were scored for temperature-sensitivity by replica plating on YPD at 25" and 37".

I n vivo labeling by [14C]lysine and [5,6-'H]uracil: Cul- tures (25 ml) of yeast (about 2 X lo7 cells/ml) exponentially growing at 25" on SD medium (supplemented with casamino acids and with 20 mg/ml adenine, trypotophan and uracil) were further cultivated for 30 min in water baths at 25" and 37". The cells were then filtered on 0.45-pm filters and transferred to the same medium except that the uracil concentration was 0.5 mg/ml. ['HIUracil (5.104 Bq/ ml) and ['4C]lysine (2.104 Bqiml) were added and the cultures were further grown for 45 min. In pulse-chase experiments, the labeling pulse was of 15 min and was chased during 30 min of by adding unlabeled uracil at 200 mg/ml. Aliquots (1 ml) were precipitated by 4 ml 10% trichloracetic acid, filtered on 0.45-prn filters, thoroughly washed with 5% trichloracetic acid and counted by scintil- lation for acid-precipitable 14C and 'H activity. The incorporation of 'H in small RNA (5.8S, 5s and tRNA) was measured by extracting total RNA as follows. Cells resuspended in 5 ml of extraction buffer (50 mM Tris-HC1 pH 7.4, 10 mM EDTA, 0.10 M NaCl and 5% sodium dodecyl sulfate) were crushed in an Eaton press and extracted twice with 5 ml of a phenol-chloroform-isoamyl alcohol mixture (24:24: 1). The nucleic acids of the aqueous phase were precipated by ethanol and resuspended in 100 to 200 pI of water. Aliquots corresponding to 5 to 20 pg RNA were vacuum dried, dissolved in 20 pl of 90% formamide and run on a 6% polyacrylamide gel containing 7 M urea in 0.09 M Tris-Borate (pH 8.3) with 2.5 M EDTA for 90 min at 200 V. The gel was fixed with 7% acetic acid, immersed for 30 min in Amplifier T M (Amersham) and dried at 80" under vacuum. Labeled RNA bands were detected by autoradiography using a kodak X-Omat S film, and were quantified by densitometry.

Preparation of cell-free extracts and in vitro assay for specific transcription of the tRNA genes: Cultures (5-liter) of strains 20B-12 (pep4-3, t rpl) , D25-13A (rpc160-31, pep4-3, t rpl) and D27-7C (rpc160-41, pep4::HZS? + ) were grown on YPD and collected by centrifugation at an optical density of 2.0 at 600 nm, yielding about 30 g of wet cells.

E E E E E E E B B

I 1 1 I I I I1 n n n n

K K s

iFi pspl

i-1 yIp32-

URA 3

C E E St E

L I PSP4 1 1 B B S

FIGURE I.-Simplified restriction map of the 18.4-kb region spanning RPCl60. The black arrow corresponds to the coding sequence of RPCl60 (ALLISON et al. 1985). Endonuclease cleavage sites are symbolized by B (BamHI), K (KfinI), H (HindIII), E (EcoRI), S (SalI), X (XbaI), St (StuI) and C (ClaI). The inserts corresponding to four plasmids (pSP1, YIp32-1, YRp7-160 and pSP4) used in this study are drawn to scale in the lower part of the figure.

Cells were washed with distilled water and with the disruption buffer (200 mM Tris-HC1 pH 8.0, 1 mM EDTA, 10 mM mgC12, 10 mM 2-mercaptoethanol, 10% v/v glycerol, 4 mM phenylmethylsulfonylfluoride) and were finally resuspended in 40 ml of that buffer. After disruption in a Manton-Gaulin homogenizer, the ionic strength was ad- justed to 300 mM ammonium sulfate. The cell lysate was left 15 min on ice and centrifuged for 90 min at 35,000 rpm. The transcription assay was done in a final volume of 40 pl, in the presence of the reaction mixture which contained 40 mM HEPES pH 7.8,70 mM KCl, 5 mM MgC12, 10 mM dithiotreitol, 0.1 mM EDTA, 10 v/v glycerol, 3 mM ATP, 3 mM GTP, 3 mM CTP, 0.15 mM ["PIUTP (6.7 lo3 Bq/nmol), 0.1 pg of the plasmid p02 containing the yeast tRNA?'" gene and varying concentrations of cell-free extract. Incubation was at 25" for 45 min. The synthesized RNA was separated by electrophoresis on a 6% polyacrylamide gel in the presence of 7 M urea and subjected to autoradiography as described above.

RESULTS

Cloning and genetic localization of a 18.4-kb region spanning gene RPCl60 on chromosome X V : The 2.5-kb EcoRI fragment of RPClGO described by RIVA et al. (1986) corresponds to about the first half of the coding sequence (Figure 1 and ALLISON et al. 1985). To isolate the whole gene, this fragment was used to probe a genomic library from strain X2 18OC of S . cerevisiae on phage A EMBL3 (M. SNYDER, unpublished data). Two partly overlapping clones were analyzed by restriction mapping using 18 dif- ferent enzymes. They define a 18.4-kb region which bears the whole gene. A simplified restriction map is given Figure 1 (a more detailed map is available on request).


The 3.6-kp EcoRI fragment containing the proximal part of RPCl60 interrupted by a 1.1-kb URA3 +

insert (Figure 1) was subsituted for one of the two wild-type alleles of RPCl60 in the ura3-52 diploid strain CMY214. In this experiment, the yeast cells were transformed by the linearized EcoRI fragment and URA3 + transformants were selected on the appropriate medium (ROTHSTEIN 1983). One transformant (strain T2) was further analyzed by hybridization of its genomic DNA after EcoRI digestion, using the 2.5-kb EcoRI fragment of RPCl6O as ra- dioactive probe. In the untransformed strain, this hybridization revealed only the 2.5-kb band corresponding to the two RPCl60+ alleles, whereas an additional 3.6-kb band corresponding to the interrupted rpcl60:: URA3 + allele was present in strain T2 (data not shown). Fourteen tetrads were dissected after sporulation of T2. All showed a 2 : 2 segregation of two viable spores (auxotrophic for uracil) and two lethal spores, as expected if the rpcl60 : : URA3 + allele were haplolethal and in agreement with equivalent results of INCLES et al. (1984). Since the spore lethality observed could reflect a defective germination rather than a failure to undergo vegetative growth, we constructed the haploid strain T12-2B (pSP4) which carries rpcl60 : : URA3 + as a chromosomal marker but is viable because it also contains a functional RPClGO gene on the centromeric plasmid pSP4 (CEN3, LYS2+, RPCl6O+). T12-2B was cultivated for about ten generations on liquid YPD, plated on YPD and about 2 X 10' colonies were examined for spontaneous plasmid loss leading to lysine auxotrophy. No auxotrophic colony was detected, whereas a control experiment with a RPCl6O + strain carrying pSP4 yielded about 5% auxotrophs. This indicates that pSP4 cannot be lost in a rpcl60 : : URA3 + background, showing that the RPCl60 + allele is indeed essential to the viability of vegetatively growing cells.

To map RPCl60' to a chromosome, an rpcl60 :: URA3 + allele was constructed by the technique described above in three diploid strains (D8, D9 and D10) which are homozygous for ura3-52 and spol l , but heterozygous for a variety of genetic markers located on each of the 16 chromosomes identified in S . cerevisiae (Table 1). The spoll mutation eliminates meiotic crossing-over without completely preventing the production of viable haploid spores in the meiotic offspring, which provides a simple chromosome allocation technique (KLAPHOLZ and EASTON-ESPOSITO 1982). In this particular case, the only viable haploid spores are those harboring the RPCl60 + allele which are consequently auxotrophic for uracil. Since there is no meiotic crossing over, the RPCl60+ allele present in these viable spores should cosegregate with the genetic markers present in cis on the same chromosome, and should show no linkage with the markers located on other

TABLE 2

Localization of rpcl60 on the right arm of chromosome XV by tetrad analysis

Segregation of the genetic markers

RPCl60 RPCl60 tsm8740

tsm8740 ade2 ade2 X X X

P T N P T N P T N

Our data 68 4 0 142 21 0 59 13 0 HILGER, PR~VOT 38 13 0

and MORTIMER

Pooled data 97 26 0

Average map 2.8 (1.2-7.3) 6.4 (3.0-9.8) 10.6 (7.0-14.2) distances

Map distances are given in centimorgans (% of recombinants) with the confidence interval at 95% given in parentheses. P stands for parental ditypes, T for tetratypes and N for nonparental ditypes. Confidence intervals were calculated assuming a Poisson distribution. Average map distances were calculated on data pooled from several crosses using either ts alleles or a small duplication of RPCl60 (see RESULTS) and including 41 tetrads analyzed by HILGER, PR~VOT and MORTIMER (1982), thus ignoring possible heterogeneities due to differences between the genetic back- grounds.

chromosomes. About 100 viable haploid segregants were isolated from each of the transformed diploids as described by KLAPHOLZ and EASTON-ESPOSITO (1982). The mutant and wild-type allele of each marker segregated at roughly equal frequencies in the offspring of the diploid transformants TD8, TD9 and TD10, except for ade2 and pet1 7 which are both located on chromosome XV. For these two markers, the offspring were either 100% wild type or 100% mutant, depending on the diploid strain considered. This indicates that RPCl60 itself is located on chromosome XV.

T o precisely localize RPCl6O on chromosome XV, we have constructed the haploid strain T4-1 (ura3- 52, his3-A200, ade2-101) carrying the haplolethal allele rpcl60:: URA3 + and the wild-type allele RPCl60+ in tandem duplication. In this strain, RPCl60 is genetically marked by the URA3 + insert but the haploid mutant is viable because of the adjacent RPCl60 + allele. Preliminary results indicated that RPCl6O may be meiotically linked with ADE2. This prompted us to cross T4-1 to an ura3-52 tsm 8740 partner. This cross is heterozygous for RPC160, ADE2, TSM 8740 and HIS3 which are all on chromosome XV, with TSM8740 close to ADE2. The segregation of RPCl6O was followed by the segregation of the URA3 + character. Seventy-two tetrads were analyzed: 55 were parental for RPCl60, ADE2 and TSM8740. Four had undergone a crossing- over between RPCl6O and TSM8740, but remained parental for the RPCl60-ADE2 interval. The remain- ing 13 had undergone a crossing over between ADE2 and RPCl60 but remained parental for the RPCIGO-

RNA Polymerase Mutants in Yeast 52 1

and FRIESEN 1987; NONET et al. 1987). As sumarized in Figure 2, a 5.3-kbXbaI fragment of RPCl60 lacking the last 386 nucleotides of the C-terminal end but including the N-terminal end was prepared as single- stranded DNA from plasmid YIp32-1 (URA3 +), tak- ing advantage of the f l origin of replication harbored on that plasmid (BALDARI and CESARENI 1985). This DNA was treated with NaHS03 and amplified by transformation in an ung strain of E . coli to generate a pool of about 2000 clones of mutagenized double- stranded plasmids. The mutagenized plasmids were integrated into the genome of the ura3-52 yeast strain YNN281 by transformation and targeted crossing- over (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981) of RPC160 at the unique ClaI site. This generated a tandem duplication of RPCl60, with one inactive copy lacking the C-terminal end and one functional copy in which the first 3.7 kb of the coding sequence ( i . e , , the region proximal to ClaI) arose from the mutagenized plasmid. A plasmid-born mutation will therefore be phenotypically expressed upon its genomic integration provided that it is located upstream of the ClaI site so as to recombine into the functional copy of RPCl60. Mutant that had excised the URA3 +

plasmid from the chromosome by a spontaneous crossover between the two direct repeats of RPC160 were selected as described by BOEKE, LACROUTE and FINK (1984). Provided that the excisive crossing over was located upstream of the ts mutation in RPCl60, this mutation will remain on the chromosomal copy of RPC160 after plasmid excision. The final result is a precise substitution of the wild-type RPCl60 gene by a mutagenized RPCl60 fragment (Figure 2).

From about 5000 URA3 + transformants isolated after 1 week of incubation at 25", six showed little or no growth when replica plated on YPD at 37" but grew at 25". The temperature-sensitive character was not due to uracil auxotrophy. Tetrad analysis from a cross against the ura3 tester strain NC93SP1 showed that, for two mutants, the ts character segregated independently from the URA3 + marker and was therefore not born by the integrated plasmid. How- ever, genetic evidence establishes that the four re- maining ts mutations are located on the integrated YIp32-1 plasmid (Table 3). (1) They are recessive, have a 2 : 2 segregation, cosegregate with URA3 + and are strongly linked to ade2. (2) Spontaneous plasmid excision strongly correlates with a reversion of the ts character. (3) Conversely, reversion of the ts mutation was very frequent (between 5 X and lop3 per cell) and was often but not always accompanied by plasmid excision. Reversion without plasmid excision was ascribed to intergenic conversion or unequal crossing over between the two direct repeats of RPCI6O (KLEIN and PETES 1981). (4) For each transformant, we obtained spontaneous subclones which

URA 3

w I

I,

t.rg.1.d Intogntlon

U R A 3 5' 3'

C l d l Cla I I

1 - 5' 3'

mutrnt 811010 Ch10mOWm8l b C U S

FIGURE 2.-Construction fo chromosomal mutant alleles of RPC160 by integrative crossover with a plasmid-born RPC160 insert. A single-stranded preparation of plasmid YIp32-1 containing an XbaI insert of RPC160 lacking the 3' end was mutagenized with NaHSOs and amplified in an ung E . coli strain to generate a pool of double-stranded mutant plasmids. Plasmid integration by targeted crossover (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981) at the ClaI site generates a tandem duplication of RPC160 with one truncated and one complete RPC160 sequence. Potential ts mutations [symbolized by (*)I present on the plasmid are trans- mitted to the nontruncated RPC160 copy provided that they are located upstream of the ClaI site (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981; SHORTLE, NOVICK and BOTSTEIN 1984). The resulting transformant should therefore express the ts phenotype. Spontaneous excision of the plasmid by an intergenic crossover between the two RPCIGO repeats restores a single copy of RPC160 which harbors the ts mutation provided that the excisive crossing over occured upstream of the mutated site.

TMS8740 interval. Ninety-one tetrads from various two-factor crosses between ADE2 and the ts alleles of RPCI6O (see below) have also been analyzed. These data, sumarized in Table 2, allow a precise localization of RPCl60 on chromosome XV, between TSM 8740 and ADE2, only 3 cM from the former marker (Table 2). The tsm8740 mutation is not complemented upon transformation by the centromeric LYS2 +, RPCl60 + plasmid pSP4 and is therefore not allelic to RPCl60.

Construction of temperature-sensitive mutant alleles of RPC160 by random mutagenesis in vitro: Temperature-sensitive (ts) mutant alleles of RPCl60 were isolated by an approach previously used to obtain ts mutants for the genes encoding actin, topoisomerase I1 or the largest subunit of RNA polymerase B in S. cerevisiae (SHORTLE, NOVICK and BOT- STEIN 1984; HOLM et al. 1985; HIMMELFARB, SIMPSON


TABLE 3

Properties of the ts mutants

Doubling time on Meiotic linkage' Mitotic instability YPD

(in hr)" URA3 + a d d Fre uency (%) of ~~ Frequency of Jasmid loss

reversion after among the LS+

25" 37" P T N P T N plasmid loss revertants' Strain

YNN281-15 (rpcl60-15) 4.0 15 1 6 0 0 1 5 1 0 22125 17 YNN281-20 (96160-20) 3.5 8.8 16 1* 0 16 1 0 2 1/25

4.3 >20 26

YNN281-31 (rp~160-31) 8 0 0 8 0 0 2 1/25 4.5 >20

62 YNN281-41 (rpc160-41) 1 5 0 0 1 2 3 0 24/25 29

a YNN288: 2.5 hr (25") and 2.7 hr (37"). ' P, T and N as defined in Table 2. The lJRA3+ character allows to follow the segregation of the chromosomally integrated plasmid bearing the ts mutation in the original construction. Tetrad analysis was based on asci with four or three viable spores. The tetrad labeled with (*) and classified as "tetratype" corresponded to an ascus where one of the two temperature resistant spore was prototrophic for uracil (ie., harbored plasmid YiP32-1). This segregant may have resulted from a spontaneous reversion of the ts allele without concomitant plasmid excision, possibly due to intergenic conversion (KLEIN and PETES 1981). ~ Average of thiee experiments on set of 50 revertants each.

had excised the plasmid but remained ts (due to a crossover localized upstream of the ts mutation). As expected, the ts mutation was much more stable in these subclones (less than one revertant in lo6 cells), since intergenic conversion or unequal crossing over would no longer be possible with only one copy of RPCl6O left on the chromosome.

These constructions yielded four recessive mutant alleles (rpcl60-15, rpcl60-20, rpcl60-31 and rpcl60- 41 ) that result from the precise substitution of the wild-type RPCl60 + gene by the mutated gene. In contrast to the initial transformants, they are genetically stable and harbor no foreign DNA sequence (originating from the transforming vector) in the vicinity of RPCl6O. Admittedly, the ts mutations could map to a gene fragment immediately proximal to RPCl6O on the mutagenized insert. To eliminate this possibility, we established that the four rpcl60 mutants define a single complementation group by mating them with a rpcl60-41 segregant and showing that the resulting diploids are temperature-sensitive. We then crossed a lys2-801, rpcl60-15 mutant to strain T12-2B (pSP4) which is rpcl60: : URA3 + but is viable because it harbors the centromeric plasmid pSP4 (LYS2 +, RPC160+) . The resulting rpcl60 : : URA3 +lrpc160-15 (pSP4) diploid was temperature-resistant because of the RPCl60 + allele of pSP4. However, spontaneous curing of the plasmid (leading to lysine auxotrophy) yielded a ts phenotype, indicating that rpcl60-15 is not complemented by rpcl60 : : URA3 + and therefore that these mutations define the same gene.

Phenotype characterization of the ts mutants: The ts mutants hardly grow at 37" and have an about twofold reduced growth rate at 25" as compared to the RPCl60+ strain YNN281. Upon a temperature shift, growth (as measured by turbidometry at 600 nm) only became arrested after three to four cell

generations. Minor differences in growth rates (Table 3) suggest that the mutants correspond to distinct mutations. Cell morphology was normal and the cell appeared to be arrested at various cell cycle stages as judged from the percentage of budded cells and the size of the buds. Diploid cells YNN281-41 X D27-7c (homozygous for rpcl60-41) were precultured at 25" in liquid YPD and plated on sporulation medium at the same temperature. This yielded only 5% of asci (most of them mature four-spore asci) as compared to the 45% of asci observed for the RPCl60+ diploid YNN281 X YNN282 under the same experimental conditions. Practically no asci were observed when the mutant diploid had been precultured at 30" or at 25" followed by a 16-hr shift at 37".

We have measured the incorporation of labeled uracil into tRNAs, 5s rRNA and 5.8s rRNA on wild- type, rpc160-31 and rpc160-41 cells exponentially growing at 25" and on cells shifted to 37" for 1 hr. We used either a 45imin pulse or a 15-min pulse followed by a 30-min chase with a 400-fold excess of unlabeled uracil. Under these conditions, there was no effect of the rpcl60-41 mutation on protein synthesis and on total RNA synthesis as measured by the incorporation of [14C]lysine or [3H]uracil in acid precipitable material (data not shown). The incorporation of [3H]uracil into tRNAs, 5s rRNA and 5.8s rRNA after the 45-min pulse was measured by autoradiography and quantified by densitometry (Fig- ure 3). As compared to wild type, the accumulation of tRNAs was reduced about five times in the rpcl60- 41 mutant at 25" and was further reduced by a factor of about four in cells that had been shifted to 37" for 1 hr or 16 hr. There was no effect on 5.8s rRNA accumulation at 25" or 37" and, more surprisingly, little effect on 5s rRNA accumulation at 25" or at 37" after 1 hr, although the latter rRNA is known to be synthesized by RNA polymerase C. The 5s rRNA

RNA Polymerase Mutants in Yeast 323 hu I[ ~ A llill M B

L A W T(16-c) 41 (irh at 37'C)

0 * a A n # -

k A L k J , 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

FIGLRE 4."In vitro transcription of the yeast tRNA$" gene in cell-free extracts from the RPC160 + strain D20B- 12 and from the

41 (th .t 37'C) rpc160-31 strain D25-13A collected from log phase cultures on YPD at 25". Panel A: 5 , 15, 25 and 35 pg of protein from wild- 4v (15.c)

FIGURE 3.-Incorporation of ['HH-5,6]uracil into small-sized RNA after 45 min of in vivo labeling at 25" and at 37" in strain YNN281 ( R P C 1 6 0 + ) and D27-7C (rpc160-41). The RNA was prepared, separated on a polyacrylamide gel and autoradi- ographed as described in YATERIALS ASD METHODS. Autoradi- ograms were quantified by densitometry. Left panel: wild type (top) and mutant (bottom) from exponential cultures at 25". Right panel: mutant cells shifted to 37" for 16 hr (top) or 1 hr (bottom).

synthesis was strongly reduced in cells that had been transferred for 16 hr at 37". Similar results were obtained after a 15-min pulse with and without chase, indicating that the 5.8s rRNA, 3s rRNA, and rRNA are very stable in the mutant and wild-type cells (data not shown). Therefore, the differences observed on the accumulation of tRNA and 5 s rRNA in the mutant cells is likely to be entirely due to differences in the rate of synthesis of these molecules. Similar results were obtained with the rpcl60-31 mutant D25- 13A (data not shown).

RNA polymerase C activity in cell-free extracts: Two mutants (rpc160-31 and rpcl60-41) were tested for the activity of RNA polymerase C in cell-free extracts of cells exponentially growing at 25", using a specific assay for transcription of tRNA genes. Figure 4 shows that the transcription activity of a cell-free extract from a rpcl60-31 strain was strongly reduced as compared to RPC160' (a similar but somewhat less marked effect was observed with rpcl60-41; data not shown). Mixing experiments showed that this reduced activity was not due to the presence of an inhibitor of transcription in the mutant extract (Figure 4, lanes 9-12). The assay was done at 25", but similar results were obtained when the extract was incubated at 37", thus showing no marked temperature sensitivity of the mutant enzyme under these experimental conditions (data not shown).

type cell-free extract (lanes 1-4) and a mutant cell free extract (lanes 5-8) were assayed for specific transcription of the tRNA"'" gene on plasmid p02 at 25". The RNA synthesized was separated on a 6% polyacrylamide gel (see MATERIALS ASD METHODS). The arrow points to the tRNA band. Panel B: the same assay as done in the presence of 20 pg of wild-type proteins (lane 9), of 20 kg of mutant proteins (lane lo), of a mixture of both (lane 11) and of 40 pg of wild-type proteins (lane 12).

Selection of an extragenic suppressor isolated from the rpcl60-41 strain: An exponential culture of strain YNN281-41 (rpcl60-41) was UV mutagenized to about 30% survival and plated on YPD at 37". Colonies growing at that temperature appeared after 1 week at a frequency of about 1 in 10' viable cells. Ten of them were reisolated and their doubling time on YPD was measured at 37". Three grew as rapidly as a wild-type strain and gave no ts segregants when backcrossed to the RPCI6O' strain YNN282. They are likely to result from a reversion of rpcl60-41 to RPCl60 ' . Out of seven others that grew rather slowly on YPD at 37" (doubling times of 360-400 min), one particular revertant (Y NN28 1-4 1-3) was backcrossed to the rpcl60-41 strain D27-7C and to YNN282 (RPC160 '). Tetrad analysis showed that the capacity to grow at 37" had a monogenic (2:2) segregation in the former cross, and that when YNN281-41-3 was crossed to YNN282, the ts phenotype was recovered in only 25% of the spores. These results indicated that the capacity to grow at 37" is due to a monogenic suppressor of rpcl60-41 which is genetically unlinked to RPC160. The corresponding mutation has been called pcil-3 (for polymerase C interaction). The growth rate of a pcil-3 RPC160' segregant on YPD at 25" and 37" is indistinguishable from wild type showing that the pcil-3 mutation alone has no detectable growth phenotype. This segregant could be


identified by its capacity to restore the original suppressor phenotype when crossed into a rpc160-41 background.

DISCUSSION

Previous work (INGLES et al. 1984; ALLISON et al. 1985; RIVA et al. 1986; THURIAUX et al. 1986) has identified RPC160 as a unique gene encoding the largest subunit of RNA polymerase C. This gene has a marked homology to the ones encoding the p' subunit of E. coli RNA polymerase (ALLISON et al. 1985) or the large subunit of RNA polymerase A and B in yeast (ALLISON et al. 1985; RIVA et al. 1986). R P C l 6 0 maps on the right arm of chromosome XV, close but not allelic to TSM8740, a gene encoding a presumably essential but hitherto unidentified function (HILGER, PREVOT and MORTIMER, 1982). SPO- T I 4 [which is involved with sporulation (TSUBOI 1983)] also maps very close to TSM8740 (possibly between it and R P C l 6 0 ) and may even be allelic to RPC160 since homozygous rpcl60 diploids have a strongly reduced level of sporulation at 25". RIVA et al. (1986) have cloned 22 other genes potentially encoding various subunits of RNA polymerase A, B and C. Their restriction maps show no overlap with the 18.4-kb insert cloned in the present work (our unpublished results). Thus, our data do not so far suggest any remarkable feature in the organization of the surrounding region on the chromosome, and certainly argue against a cluster of genes encoding RNA polymerase subunits.

Temperature-sensitive rpcl60 mutants were obtained by in vitro mutagenesis with NaHS03 on a non-replicative plasmid which was subsequently inserted into the chromosome by integrative transformation (SHORTLE, NOVICK and BOTSTEIN 1984). This procedure is somewhat more cumbersome than an alternative approach based on plasmid exchange (MANN et al. 1987; BUDD and CAMPBELL 1987) but has the advantage of yielding mutations which have been precisely substituted for the wild-type allele on the chromosome, rather than yielding mutations born on an artificial extrachromosomal replicon. As al- ready observed (SHORTLE, NOVICK and BOTSTEIN 1984) ts mutants unlinked to the mutagenized gene were also generated (2 out of 5000 transformants). One explanation for the unlinked ts mutants is that integrative transformation is restricted to the small cell subpopulation which is competent for mitotic recombination (HENACT and LUZZATI 1972; MINET, GROSSENBACHER-GRUNDER and THURIAUX 1980) and which may also be prone to a high level of spontaneous mutation. However, we observed no mutagenic effect of transformation on spontaneous mutation as monitored by resistance to canavanine. In addition to the unlinked ts mutants, four ts alleles of RPClGO have been identified by their cosegregation with the

integrated plasmid, their close linkage to ADE2 on chromosome XV, their frequent reversion upon spontaneous plasmid excision and their failure to complement a rpcl60: : URA3 + null allele. These four alleles may be due to distinct mutations as judged from differences in their reversion patterns and in their growth rates at 25" or 37".

The two ts mutants analyzed in some detail (rpcl60- 31 and rpc160-41) are defective in tRNA synthesis both in vivo and in cell-free extracts. The synthesis of 5.8s rRNA catalyzed by RNA polymerase A was not affected. As anticipated, fully defective rpcl60 mutants are unable to undergo spore germination (INGLES et al. 1984) and vegetative growth (this work). Yet, the ts mutant rpcl60-41 which have a fivefold reduction in tRNA synthesis in vivo at 25" has a less than twofold reduction in its growth rate on rich medium at the same temperature, indicating that tRNA synthesis (or at least its first step, which is the transcription of the corresponding genes by RNA polymerase C) is not a major rate limiting factor in cellular growth.

Temperature-sensitive mutants need not be ther- mosensitive for the activity of the corresponding enzyme, but may instead affect its synthesis (or its stability), especially in the case of complex multimeric enzymes such as RNA polymerases. Several ts mutants defective in the assembly of RNA polymerase have indeed been described in E. coli (KIRSCHBRAUM et al. 1975). In yeast, MANN et al. (1987) have isolated plasmid-born ts alleles of RPC40, the gene encoding the AC 40 subunit common to RNA polymerase A and C. The corresponding mutations prevent the in vivo assembly of RNA polymerase A and C but do not affect the activity of the preassembled enzyme. A similar conclusion has been reached forts mutations in the gene encoding the largest subunit of RNA polymerase B in S. cerevisiae (HIMMELFARB, SIMPSON and FRIESEN 1987). In contrast, the mutation isolated in the latter gene by NONET et al. (1987) appears to affect the activity rather than the intracellular accumulation of the enzyme, but completely lacks RNA polymerase B activity in vitro even from cells grown at the permissive temperature.

In the present case, the two ts mutants analyzed show an additional fourfold decrease in tRNA synthesis in vivo after a one hour shift to 37". Thus, it seems unlikely that the mutations merely prevent the assembly of RNA polymerase C without affecting the properties of the enzyme present before the temperature shift. In the latter case, the preexisting RNA polymerase molecules would continue to produce RNA for several hours before becoming limiting, assuming that RNA polymerase C is not an unusually labile enzyme. However, we cannot exclude that the mutation itself has a temperature-sensitive effect on the stability of the enzyme (alone or associated with

RNA Polymerase Mutants in Yeast 525

its cognate transcription complexes) rather than a direct effect on its transcriptional activity. In particular, this hypothesis would explain that the reduced RNA polymerase C activity of the mutant cell-free extracts is not particularly temperature-sensitive.

A striking feature of the two mutants analyzed is that they are largely specific for the synthesis of tRNA, although the synthesis of 5s rRNA eventually became arrested after three doubling times at 37”. This indicates that the mutant enzyme (and, possibly, the wild type form as well) discriminates between the structurally related initiation complexes formed between the tRNA and 5s rRNA genes and several proteins acting as transcription factors (SENTENAC 1985). One possibility is that the mutationally modi- fied C160 subunit has a specifically altered recogni- tion site for the initiation complexes. Alternatively, the affinity of the wild type RNA polymerase C could be lower for tRNA initiation complexes, which would account for the reduced synthesis of tRNAs if the ts mutations lead to a decreased intracellular concentration of the enzyme.

Within the ribosomal repeat unit, the 5s rRNA gene lies between the major promoter element and the initiation site of the large rRNA gene, and is transcribed in opposite orientation (ELION and WAR- NER 1984). This suggests that the transcription of the 5s rRNA gene by RNA polymerase C may be required for the synthesis of the 35s rRNA by RNA polymerase A, thus ensuring the coordinate production of both types of rRNA. However, our data establish that the synthesis of 5.8s rRNA (and, consequently, of the 35s rRNA) is not affected by a severe reduction in the rate of 5s rRNA synthesis. Therefore, the pe- culiar organization of the rRNA repeat unit in S . cerevisiae does not result in a tight coordination of the transcription of the rDNA genes by RNA polymerases A and C.

In conclusion, the present work provides a first characterization of conditional mutants specifically defective in RNA polymerase C. The properties of the mutant RNA polymerase C enzymes are being analyzed biochemically in order to identify the molecular defect which leads to their temperature-sensitive phenotype, together with a parallel study on gene RPC40 (MANN et al. 1987). Yeast genes corresponding to other subunits of RNA polymerase C have been cloned by immunological screening (RIVA et al. 1986) and are currently being characterized in this laboratory. Still other genes related to RNA polymerase C function may be identified by selecting for extragenic suppressors of rpcl60 ts mutants. In- deed, one of the ts alleles described here (rpcl60-41) is suppressible by an extragenic suppressor mutation, pci l -3 , which has by itself no detectable growth phenotype. We have no indication as to the molecular mechanism of this suppression, but one obvious

possibility is that pczl-3 encodes a protein directly interacting with the C160 subunit, for example a transcription factor or another subunit of the polymerase.

We thank CARL MANN and PIERRE FROMAGEOT for stimulating discussions and encouragement. This work was partly supported by a long term EMBO Fellowship to R.G. and by a grant from the Centre National de la Recherche Scientifique (ATP 033-542).

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Conditional Mutants of RPCl60, the Gene Encoding the ... · transformation of the E. coli strain BW310, about 2000 pooled ampicillin-resistant transformants were harvested in LB supplemented