The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σ^A-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^A-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^A-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^A-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^A that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

枯草芽孢杆菌的调节性 6S-1 和 6S-2 RNA以亚微摩尔亲和力与管家 RNA 聚合酶全酶 (σ ^A -RNAP) 结合。我们观察到来自已发表的表达 His 标记 RNAP 的枯草芽孢杆菌菌株的内源 6S RNA 的共纯化。^{σ A} -RNAP 制剂中的此类 6S RNA 污染减少了可与 DNA 启动子结合的酶的比例。此外，这导致通过 σ ^A -RNAP 利用共纯化 6S RNA 作为模板合成称为产物 RNA (pRNA) 的短流产转录物的背景 RNA 合成。为了避免这个问题，我们构建了一个B. subtilis菌株表达带His标记的RNAP但携带两个6S RNA基因的缺失。His-tagged、6S RNA-free σ ^A -RNAP 全酶可以通过一个亲和步骤制备，具有足够的纯度和活性。我们还报告了枯草芽孢杆菌σ ^A的表达和单独纯化，可以将其添加到带有 His 标记的 RNAP 中，以最大限度地提高全酶的量，并通过推断，体外转录活性。