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

Preparations of Bacillus 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 T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit [Jaehning, J. A., Wiggs, J. L. & Chamberlin, M. J. (1979) Proc. Natl. Acad. Sci. USA 76, 5470-5474]. By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have 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 dictated by a peptide of Mr 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. Hence the 28,000 Mr 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.     

An Immunological Assay for the Sigma Subunit of RNA Polymerase in Extracts of Vegetative and Sporulating Bacillus subtilis

The activity of the sigma subunit of Bacillus subtilis RNA polymerase decreases markedly during the first hours of sporulation [T.G. Linn et al. (1973) Proc. Nat. Acad. Sci. USA 70, 1865-1869]. We have prepared antibody against RNA polymerase holoenzyme to determine the fate of sigma polypeptide during spore formation. This antiserum specifically and independently precipitates sigma and core polymerase from crude extracts of B. subtilis as judged by both sodium dodecyl sulfate and urea gel electrophoresis of the precipitates. We report that crude extracts of sporulating cells lacking sigma activity contain as much sigma polypeptide as extracts of vegetative cells. However, sigma polypeptide in extracts from sporulating cells is apparently only weakly associated with RNA polymerase, as indicated by the failure of sigma to co-purify efficiently with core enzyme during phase partitioning.The loss of sigma activity and the weak binding of sigma to core enzyme occurs normally in a mutant blocked at an intermediate stage of sporulation (SpoII-4Z) and in wild-type bacteria sporulating in 121B medium, Difco sporulation medium, or Sterlini-Mandelstam resuspension medium. In contrast, sigma in two mutants (SpoOa-5NA and SpoOb-6Z) blocked at an early stage of spore formation remains active and tightly associated with RNA polymerase during stationary phase.     

Altering the interaction between sigma70 and RNA polymerase generates complexes with distinct transcription-elongation properties

We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from sigma70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between sigma70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of sigma70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and sigma70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase.