Figure 2.

Binding of σ⁵⁴ to E.coli core RNAP. Native gel holoenzyme assembly assays were used to detect complexes forming between core RNAP and R383K and R383A, respectively. The formation of holoenzyme (Eσ⁵⁴) was detected as the presence of a faster migrating species when compared with core (E, lane 1) alone. Titrations of core RNAP with σ⁵⁴ were carried out using 250 nM core RNAP and increasing concentrations of σ⁵⁴ at ratios of 1:1 (lanes 2, 5 and 10), 1:2 (lanes 3, 6 and 11), 1:4 (lanes 4, 7 and 12) and 1:8 (lanes 8 and 13). Wild-type σ⁵⁴ shifted nearly all the core into the holoenzyme form at a 1:1 molar ratio of core to σ⁵⁴ (lane 2); in contrast, R383K shifted all the core to the holoenzyme form at a ratio of 1:2 (lane 6) and R383A at 1:4 (lane 12). Free σ⁵⁴ (2.5 µM) proteins are also shown: lane 14, wild-type; lane 15, R383K; lane 16, R383A.