Figure 2.

Purified RNAP and immunoblot analysis. (A) (Left) Cellular extracts of wild-type (wt) or β′;::σ⁷⁰ (F) strains (RL301 and RL1094) were separated by 3%-8% Tris-Acetate (Novex), transferred to nitrocellulose, and probed with a mixture of β′ and σ⁷⁰ antibodies (Materials and Methods). Equal amounts of cellular protein were present in each lane. (Center) Western blot of wild-type (wt) and β′;::σ⁷⁰ (F) RNAPs. Samples were separated by 3%-8% Tris Acetate (Novex) and probed as in the left panel. (Right) β′;::σ⁷⁰ RNAP (F) purified from the strain containing the rpoC;::rpoD fusion and Trp-repressed rpoD (RL1094) separated by 4%-15% PAGE gel (Pharmacia) shown next to wild-type (wt) RNAP for size comparison. (B) β′;::σ⁷⁰ (F) or wild-type (wt) RNAPs were mixed with λP_R template, allowing the formation of open complexes at 37°C for 20 min (heparin was present where indicated for the last 10 min), separated by native gel electrophoresis (4% NuSieve agarose, 0.5× TBE), and stained with EtBr. The absence of heparin masked the slightly faster mobility of β′;::σ⁷⁰ OCs (see legend to Fig. 4C) because β′;::σ⁷⁰ OCs appeared to aggregate more than wild-type OCs.