Simultaneous Gain and Loss of Functions Caused by a Single Amino Acid Substitution in the / 3 Subunit of Escherichia coli RNA Polymerase : Suppression of nusA and rho Mutations and Conditional Lethality

Transcript elongation and termination in Escherichia coli is modulated, in part, by the nusA gene product, an acidic protein that interacts not only with RNA polymerase itself but also with ancillary factors, namely the host termination protein Rho and phage X antitermination protein, N. The E. coli nusAl mutant fails to support X development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant ( r i f R ) variants of the nusAl host support X growth. We report here the isolation and pleiotropic properties of one such .ifR mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase 0 subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42". Pulse-labeling and analysis of newly synthesized proteins indicate that the mutant cell is proficient in RNA synthesis at high temperature. Apparently, ts8 causes a loss of some specialized function of RNA polymerase without a gross defect in general transcription activities. ts8 is an allele-specific suppressor of nusAI. It does not suppress nusAsa1, nusB5 and nusE7I mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and X growth. A mutation in the N gene, punAI, that restores X growth in the nusAI mutant host but not in the nusAsuZ host, compensates for the nusAsal allele in the ts8 mutant. This combined effect of two allele-specific suppressors suggests that they enhance some aspect of polymerase-NusA-N interaction and function. ts8 suppresses the rho15 mutation, but not the rho112 mutation, indicating that it might render RNA polymerase susceptible to the action of a defective Rho protein. Marker rescue analysis has localized ts8 to a 910-bp internal segment of rpoB that encodes the Rif domain. By amplification, cloning and sequencing of this segment of the mutant chromosome we have determined that ts8 contains Phe in place of Ser522, caused by a C to T transition. By gene conversion, we have established that the simultaneous gain and loss of three functions of polymerase is caused by this single amino acid substitution. Clearly, a site in the /3 subunit critical for the functioning of both termination and antitermination factors is altered by ts8. The alteration, we imagine, might make this site on polymerase receptive to some factors but repulsive to others. I N Escherichia coli, a single RNA polymerase carries out gene transcription that consists of several major steps: (a) recognition and binding of a promoter and melting of the DNA double helix, (b) initiation of RNA chain synthesis directed by the template strand, (c) chain elongation and progression of the transcription complex along the gene, (d) recognition of the stop signal at the end of the gene and cessation of RNA chain synthesis and, finally, (e) release of the RNA from the transcription complex and dissociation of RNA polymerase from the DNA template (McCLURE 1985; VON HIPPEL et ai. 1984). RNA polymerase is a multisubunit enzyme constituted by two functional components (BURGESS 1969; BURGESS et al. 1969): (i) a core, cy2&?' (products of rpoA, rpoB and rpoC, respectively), which carries out all steps but promoter recognition and de novo initiation of RNA synthesis and (ii) one of several sigma subunits (the products of rpoD, rpoH and rpolv) that allows differGenetics 130 41 1-428 (March, 1992) entia1 promoter recognition and specific initiation (BURGESS et al. 1987; HELMANN and CHAMBERLIN 1988). Productive initiation at certain promoters, however, requires specific regulatory proteins in addition to u, which interact with both a nearby cisacting site and the polymerase to promote initiation (MCCLURE 1985). Transcript elongation and termination is modulated by the interaction of RNA polymerase with a variety of ancillary factors (FRIEDMAN 1988; YEAGER and VON HIPPEL 1987). Early during elongation in vitro, the u subunit dissociates from the transcription complex and the core enzyme then continues to elongate the RNA chain. Elongation by core polymerase proceeds at a reduced rate in vitro, as compared to in vivo. To date, a general transcription factor that might enhance the intrinsic elongation rate has not been reported. Although the core polymerase can stop RNA synthesis at some sites known as factor-independent 412 J. Sparkowski and A. Das or intrinsic terminators, additional factors are required for accurate and efficient termination at many other sites. Rho and Tau are among these factors which promote termination at distinct classes of termination sites (ROBERTS 1969; BRIAT and CHAMBERLIN 1984). The other known factor to promote termination is NusA, a 55-kD acidic polypeptide encoded by the nusA gene essential for cell viability (FRIEDMAN and BARON 1974; GREENBLATT, MCLIMONT and HANLY 198 1; KINGSTON and CHAMBERLIN 198 1; WARD and GOTTESMAN 198 1 ; NAKAMURA et al. 1987). NusA is a component of the elongating transcription apparatus (HORWITZ, LI and GREENBLATTL 1987), performing pleiotropic functions in transcript elongation and termination (see YEACER and VON HIPPEL 1987). It reduces the overall rate of elongation, promotes transcriptional pause at distinct sites and modulates the efficiency with which polymerase stops RNA synthesis at different sites. Moreover, it modulates the activity of both termination and antitermination factors (LAu, ROBERTS and Wu 1983; GREYHACK et al. 1985; WHALEN, GHOSH and DAS 1988). NusA binds not only the RNA polymerase itself (GREENBLATT and LI 198 la) but also the termination protein Rho (SCHMIDT and CHAMBERLIN 1984) encoded by the rho gene essential for viability of E. coli (DAs, COURT and ADHYA 1976; INOKO and IMAI 1976). It also binds directly to the antitermination protein N of phage X (GREENBLATT and LI 198 1 b). Thus, NusA is believed to be an adaptor or a coupler which enables various ancillary factors to interact with RNA polymerase and modulate elongation (GREENBLATT 198 1). An understanding of RNA polymerase structurefunction is central to the mechanism of gene regulation. The /3 subunit of RNA polymerase, encoded by the rpoB gene, has been implicated in most transcription steps (YURA and ISHIHAMA 1979; YEAGER and von Hippel 1987). Mutations in rpoB, readily selected by rifampicin-resistant phenotype, are known to affect gene expression and control. Certain riff" alleles alter the susceptibility of RNA polymerase to the action of the N-antitermination protein (GEORGOPOULOS 197 1; GHYSEN and PIRONIO 1972) and Rho termination protein (DAs, MERRIL and ADHYA 1978; GUARENTE and BECKWITH 1978), while others alter the intrinsic termination activity of polymerase both positively and negatively (NEFF and CHAMBERLIN 1980; YANOFSKY and HORN 1981). To gain an understanding of the mechanism of polymerase-NusA interaction and the role of this interaction in transcript elongation and termination, we have sought suppressor mutations in rpoB that compensate for a mutant nusA allele. The particular allele chosen is nusA1 which causes a specific defect in antitermination promoted by the X N gene product without grossly affecting cellular transcription and viability (FRIEDMAN and BARON 1974). Here, we report one suppressor allele, rpoBts8, caused by a single amino acid substitution in the /3 subunit residue 522, phenylalanine in place of Serine. We further demonstrate that this mutation is pleiotropic: First, in addition to allowing N-dependent antitermination with the nusAl allele specifically, the ts8 mutation (F522) also suppresses the termination defect of a mutant rho allele, rhol5. Second, in contrast to causing this gain of two opposite functions, the mutation also results in the conditional lethality. The mutant cell is unable to grow at temperatures above 40 O ; however, it does not reveal any gross defect in transcription at the nonpermissive temperature. This raises the possibility that the conditional growth arrest might result from the loss of some specialized function, for instance, interaction with a transcription factor and thereby a failure to express some essential genes. Consistent with this hypothesis, the growth defect is suppressed by a dominant mutation in a gene, we named greC, located at 90.5 min on the E. coli chromosome (SPARKOWSKI 1990). The growth defect is also suppressed by an elevated expression of yet another gene of E. coli, greA, that may encode a transcription factor (SPARKOWSKI and DAS 1990, 1991). MATERIALS AND METHODS Bacterial strains, phages and plasmids: E. coli K12 strains used in this study are listed in Table 1. XNpunAl was provided by D. FRIEDMAN (University of Michigan, Ann Arbor). Ximm2 1 c, Ximm2 1 D69, Ximm434r32, Xbzc, hcI857Nam53, XuirNamSOO and Ximm434Nam213 were obtained from the collection of S . ADHYA and R. WEISBERG (National Institutes of Health, Bethesda). Plasmid pDJJ1 containing the rpoB gene was a gift from D. JIN (University of Wisconsin, Madison). Plasmid pDL19 used to engineer rpoB fragments is a derivative of pUC 19 containing a BglII site within the multilinker (DAS 1990). Plasmid pDL34 (LAZINSKI, GRZADZIEUKA and DAS 1989) was used to supply AN protein in trans. Transformation, conjugation and other genetic manipulations and tests have followed the procedures compiled by SILHAVY, BERMAN and ENQUIST (1984). Transduction of nusA, nusB, nusE, rho and rpoB alleles was done with phage Pluir, employing linked markers Zgi::TnlO, proC::TnlO, Zhb::TnlO, ilv and argE::TnS, respectively. Prototrophs were selected by plating the transduction mixture directly on selective minimalagar plates and incubation for 2 days at the appropriate temperature. For the selection of antibiotic-resistant transductants, P1-infected cells were diluted and grown for 1 hr in Luria broth at 32" (temperaturesensitive strains and XcZts prophage strains) or 37" (all other strains) prior to plating on selective antibiotic plates. Tetracycline, kanamycin and ampicillin were used at 12.5, 25 and 50 pg/ml, respectively. Plates were incubated at 32" or 37" for 20-40 hr. Transductants obtained by lowest multiplicity of infection were purified on selective plates to single colonies and saved. Isolation of nusA2 suppressor mutants: Aliquots of 2 X 1 O9 cells of SP1 were spread on LB plates containing 20 pg/ ml rifampicin and incubated at 32" for 2 days. rip colonies (small, medium and large) were purified on the LB-Rif plates E. coli RNA Polymerase Mutant