Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Abstract

Preparations of B. subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with bacteriophage T7 DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I). Holoenzyme II lacks the normal .sigma. subunit (Jaehning et al., Holoenzyme II (1979)). By heparin-agarose chromatography holoenzyme II fractions were obtained that have no detectable holoenzyme I activity is judged by their failure to utilize promoter sites for holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template tested. These fractions are far more active with B. subtilis DNA than with T7 DNA or other heterologous templates. This high degree of specificity has allowed identification of plasmids containing cloned fragments of B. subtilis DNA that bear strong promoter sites for holoenzyme II. These promoter sites are not used at all by B. subtilis RNA polymerase holoenzyme I. The specificity of holoenzyme II is dicated by a peptide of MW 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B. subtilis core polymerase. The 28,000 MW peptide appears to be a .sigma. factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.