Effects of reduced amount of RNA polymerase sigma factor on gene expression and growth of Escherichia coli: Studies of the rpoD40 (Amber) mutation

Summary

A mutant of Escherichia coli K-12 carrying an amber mutation (rpoD40) in the structural gene for RNA polymerase σ factor and a temperature-sensitive amber suppressor (supF-Ts6) grows virtually normally at 30° C, but does not grow at 42° C due to the inability to synthesize σ polypeptides (Osawa, T. and Yura, T., Mol Gen Genet 180, 293–300, 1980). When the mutant cells are transferred from 30 to 42° C, the cellular amount of σ relative to total protein is found to decrease from 50% (at 30° C) to 10% of the wild-type level after about 2h. The decrease of σ is accompanied by a gradual decrease in RNA and protein syntheses and a sudden loss of viability. At the highest temperature (36° C) that permits steady growth of this mutant, the amount of σ and the growth rate become 6% and 50 to 60% of the wild type, respectively. These results suggest that the minimum level of σ required for growth is 0.02 to 0.04 in terms of molar ratio of σ to core enzyme, that is 6 to 10% of the wild type.

Two-dimensional gel electrophoresis of proteins synthesized under the reduced σ level reveals either markedly increased or decreased syntheses of several polypeptides, while no detectable effect is observed in the majority of polypeptides. Notably, the synthesis of a set of major heat-shock polypeptides is greatly enhanced. Hence, the decrease of RNA polymerase holoenzyme relative to the core enzyme seems to affect the synthesis of individual proteins differentially, primarily at the level of transcription. The expression of the groE operon, one of the major heat-inducible operons in E. coli is also studied in some detail.