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σB is a secondary σ factor of Bacillus subtilis. RNA polymerase containing cr transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the σB structural gene (sigB), regulate o dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the σB regulatory pathway. Evidence is now presented that RsbW inhibits σB-dependent transcription by binding to crB and blocking the formation of a σB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or σB will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-fitration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of σB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified σB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a σB-dependent promoter (ctc) in vitro. RsbW efficiently blocked σB-dependent transcription but only if it was incubated with σB prior to the addition of the core enzyme.