Figure 2.

σ-exchange assay using immobilized +16 paused complexes. (A) A scheme demonstrating the principle of ‘σ-exchange' experiments on the lacUV5 promoter-proximal pause. The pausing site (PS) is marked by an open rectangle. Promoter open complex (RPo) and paused elongation complex (TEC16) are shown. An exchange reaction between σ originated from the promoter complex (σ1 in gray) and exogenously added σ (σ2 in black) is illustrated. (B) ³²P-labeled RNA transcripts produced by σ⁷⁰-containing (lanes 1–4 and 9–11) and σ⁷⁰-less (lanes 5–8) TEC16 immobilized on Ni²⁺-NTA beads. Transcription complexes were chased by the addition of NTPs and 0.5, 1, or 5-min incubation either before (lanes 2–4) or after heparin wash (lanes 6–11). Lanes 9–11: σ⁷⁰-less complexes were supplemented with exogenous σ⁷⁰ (500 nM final concentration) before chase. (C) Crosslinking of the σ⁷⁰ subunit to ³²P-labeled lacUV5 DNA in immobilized TEC16 either before (lane 1) or after (lanes 2–5) heparin wash. Complexes were supplemented with exogenous σ⁷⁰ (lanes 3–5). NTPs were added before (lane 5) and after (lane 4) the addition of σ⁷⁰. Crosslinked σ⁷⁰–DNA complexes resolved on SDS–PAGE are shown (labeled as σ-crosslink).