RNA polymerase beta mutations have reduced sigma(70) synthesis leading to a hyper-temperature-sensitive phenotype of a sigma(70) mutant

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase, The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma(70) polypeptide is not stable at a high temperature, Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma(70) in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up, The rate of synthesis of the sigma(70) polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma(70) polypeptide after temperature shift-up was the same in both strains, We conclude that because of the reduction of expression of rpoD800 by rpoB114 in concert with the intrinsic instability of the mutant sigma(70) polypeptide, the amount of holoenzyme containing sigma(7O) becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant, Furthermore, the effect of rpoB114 on the expression of sigma(70) is independent of the rpoD800 allele and is at the transcriptional level, In vitro transcription assays shelved that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically, The effects of these rpoB mutations on gene expression are discussed.