Fig. 3.

P7 prevents RPc formation by the σ⁵⁴-containing RNAP. (a) Autoradiograph of a 4.5% (wt/vol) native polyacrylamide gel showing results from EMSA experiment with ³²P-labelled nifH promoter probe to demonstrate that P7 inhibits RPc formation by the σ⁵⁴ holoenzyme conducted as previously described [16], [30]. The components present in each lane are indicated above each image of the gel, and the schematic indicates the concentration of reaction components, time of addition, and incubation time. (b) As in (a), but the assays were conducted with Alexa488-labelled σ⁵⁴ (σ⁵⁴*) to determine the presence or absence of σ⁵⁴ in the different complexes detected in (a) by fluorescence imaging. The Alexa488-labelled version of σ⁵⁴ was prepared as described in Ref. [31]. In (a and b), the migration positions of the different protein–protein and protein–DNA complexes are indicated (see text for details). We note that we could not clearly distinguish the free σ⁵⁴* and σ⁵⁴*–³²P–nifH complex in the gels shown on the right in Fig. 3b. We explain this by suggesting that the excess of free σ⁵⁴* (800 nM) may mask the amount of σ⁵⁴*–³²P–nifH complexes formed (maximum of 10 nM) under our experimental conditions. The gels analysed by radiography were dried prior to exposure to the phosphorimaging plate, whilst gels analysed by fluorescence were not dried.