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. 1974 Jul;71(7):2872–2876. doi: 10.1073/pnas.71.7.2872

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

Robert Tjian 1, Richard Losick 1
PMCID: PMC388574  PMID: 4211798

Abstract

The activity of the σ 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 σ polypeptide during spore formation. This antiserum specifically and independently precipitates σ 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 σ activity contain as much σ polypeptide as extracts of vegetative cells. However, σ polypeptide in extracts from sporulating cells is apparently only weakly associated with RNA polymerase, as indicated by the failure of σ to co-purify efficiently with core enzyme during phase partitioning.

The loss of σ activity and the weak binding of σ 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, σ 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.

Keywords: antibody precipitation

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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