Effects of reduced amount of RNA polymerase sigma factor on gene expression and growth of Escherichia coli: studies of the rpoD450 (amber) mutation.

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