Figure 3.

Escape of the RNA polymerase from the promoter is accompanied by dissociation of σ⁵⁴ from DNA. (A) RNA polymerase subunit σ⁵⁴ is depleted from early RP_el. The experimental strategy for purification of RP_c and RP_o complexes is outlined at the top. The complexes were purified from DNA-free proteins on a Sephacryl S-400 column, separated in an SDS–PAGE and silver stained (lanes 6 and 7). Purified proteins used for preparation of the transcription complexes were of ∼95% purity and were loaded as additional markers (lanes 1–4). Total proteins present in the reaction mixture were stained with Coomassie (lane 5). In some experiments NtrC was under-represented in RP_el as compared with RP_o suggesting that NtrC was more stably bound to DNA in RP_o. NtrC was not detectable in either of the complexes if column fractionation was conducted in the presence of excess competitor DNA containing strong NtrC binding site (data not shown). M, protein molecular mass markers. (B) Functional RP_o and RP_el complexes survive Sephacryl S-400 column. RP_o and RP_el complexes were analyzed using a single-round transcription assay before (–) or after (+) fractionation on a Sephacryl S-400 column. No DNA-free proteins were added to the reaction after the column.