Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli.

Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for the sigma subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30 degrees C) but do not grow at high temperature (42 degrees C) due to the inability to synthesize sigma factor. In one mutant studied in detail (rpoD40), the rate of sigma-factor synthesis at 30 degrees C is about half that of the wild type and is decreased to 10%-15% within 1 h of incubation at 42 degrees C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in sigma synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD. supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42 degrees C) exhibit different thermostabilities attributable to alterations in the sigma factor. The reduced sigma synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as 'amber fragment'. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the sigma factor and directly affects sigma synthesis upon inactivation of the suppressor at high temperature.