Rapid preparation of 6S RNA-free B. subtilis σ-RNA polymerase and σ.

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σ-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σ-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σ-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σ-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σ that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.