Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations.
A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.
Molecular and Cell Biology Program, University of Texas, Richardson 75083-0688.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000096:biosynthesis; D012335:RNA, Ribosomal / Q000096:biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D006159:Guanosine Tetraphosphate; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase