Simultaneous gain and loss of functions caused by a single amino acid substitution in the beta 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 lambda antitermination protein, N. The E. coli nusA1 mutant fails to support lambda development due to a specific defect in N-mediated antitermination. Certain rifampicin-resistant (rifR) variants of the nusA1 host support lambda growth. We report here the isolation and pleiotropic properties of one such rifR mutant, ts8, resulting from a single amino acid substitution mutation in rpoB, the structural gene for polymerase beta subunit. ts8 is a recessive lethal mutation that blocks cell growth at 42 degrees. 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 nusA1. It does not suppress nusAsal, nusB5 and nusE71 mutations nor does it bypass the requirement for a functional N gene and the nut site for antitermination and lambda growth. A mutation in the N gene, punA1, that restores lambda growth in the nusA1 mutant host but not in the nusAsal 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 beta 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.
Sparkowski
J
J
Department of Microbiology, University of Connecticut Health Center, Farmington 06032.
Das
A
A
D000483:Alleles; D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005804:Genes, Lethal; D005808:Genes, Recessive; D008040:Genetic Linkage; D007700:Kinetics; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors; D010641:Phenotype; D012234:Rho Factor / Q000235:genetics*; D013489:Suppression, Genetic; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D012234:Rho Factor; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases