Fig. 5. Biochemical characterization of YvrI as a σ factor.

A) Purified RNAP preparations used in assays either with σ^A (Eσ^A) or depleted for σ^A (E). B) YvrI and YvrHa-dependent multi-round transcription from P_oxdC in vitro. Template DNA (10 nM) encoding P_oxdC was included with 100 nM purified B. subtilis RNA polymerase (Eσ^A) and 2 μM each of purified σ^A, YvrI, and YvrHa as indicated. Specific transcription from P_oxdC is predicted to yield an 82 nt run-off transcript. The asterisk indicates the weak signal noted in reactions supplemented with YvrI alone. C) KMnO₄ footprinting at the oxdC promoter (template strand) using 37 nM B. subtilis RNAP (E) shown in panel A or 100 nM E. coli core polymerase (Epicentre Biotechnologies) (E (Eco)). Reactions contained RNAP alone (lane 1), with YvrI (lane 2), both YvrI and YvrHa (lane 3), or YvrI, YvrHa and σ^A (lane 4) Nucleotide assignments are based upon an A/G ladder generated from the same strand (not shown).