RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant.
Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit. The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006358:Hot Temperature; D009154:Mutation; D012293:Rifampin / Q000494:pharmacology*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D012321:DNA-Directed RNA Polymerases; D012293:Rifampin