Extended Data Fig. 1: Overexpression and purification of Cdiff RNAP.

a, pXC026, overexpression plasmid for the Cdiff rpoA, rpoZ, rpoB, and rpoC genes (encoding the α, ω, β, and β′ subunits of Cdiff RNAP, respectively). The β and β′ subunits were fused with an inter-subunit 10-amino-acid (aa) linker (LARHVGGSGA) and a C-terminal Rhinovirus 3C protease-cleavable His10 tag. b, (Top) Size-exclusion chromatography profile for the assembled Cdiff RNAP Eσ^A. (Bottom) Coomassie-stained SDS-PAGE of individual fractions from major peaks. RNAP subunits are labeled on the right of the gel. The yield for Cdiff RNAP Eσ^A from pooled fractions of the second peak was sufficient from single purification and used for biochemistry and structural biology experiments. c, Abortive transcription assay with Cdiff core and Eσ^A using the Cdiff rrnC promoter as DNA template. The transcriptional activity of Cdiff Eσ^A was inhibited with increasing concentrations of Fdx. The transcription assays were repeated three times independently with similar results (n = 3). The result is shown from one representative experiment. The result is shown from one representative experiment. Lane 1, Cdiff RNAP core; lane 2, Cdiff Eσ^A; lane 3, Cdiff Eσ^A with 0.2 µM Fdx added; lane 4, Cdiff Eσ^A with 2 µM Fdx added.