Figure 5.

Probing of the interactions between σ⁷⁰ or its fragments and the-10 oligonucleotide using UV crosslinking assay. (A) Crosslinking of the −10 non-template oligonucleotide with the wild-type σ⁷⁰ (FL) or σ⁷⁰ fragments (1–2, 2b, and 2a) present at the indicated concentrations in the absence or in the presence of 20 nM of RNAP core. Crosslinked complexes were resolved by 8% SDS–PAGE. (B) Quantification of data from panel A obtained in the presence of 0.1 μM σ⁷⁰ or its fragments ‘Stimulation fold' is a ratio of crosslinking signals observed in presence and in the absence of RNAP core. Mean values and standard deviations from three independent experiments are shown. (C) Crosslinking of the non-template (NT) (lanes 1–4) and control template (T) (lanes 5–12) −10 element oligonucleotides with RNAP holoenzymes (lanes 1–8) containing either wild-type σ⁷⁰ (FL) or indicated σ⁷⁰ fragments. Lanes 9–12: crosslinking of σ⁷⁰ and σ⁷⁰ fragments without core RNAP. The samples contained 0.5 μM (lanes 1–4) or 5 μM of σ (lanes 5–12).