Fig. 2.
P7 inhibits transcription initiation by the σ54-containing RNAP. (a) An autoradiograph of a 20% (wt/vol) denaturing gel showing the synthesis of the transcript by P7SEσ54 (lanes 1–5) or WTEσ54 (lanes 7 and 8) in the absence and presence of increasing amount of P7 from the following σ54-dependent promoters: (i) nifH (transcript = UpGpGpG), (ii) glnH P2 (transcript = UpGpU), and (iii) relA P4 (transcript = CpUpGpG). All transcripts are indicated by an arrow where the underlined nucleotides are 32P labelled. P7 was added to the reactions prior to holoenzyme formation as is indicated in the schematic. All data obtained in at least three independent experiments fell within 5% of the relative %A value shown. (b) An autoradiograph of a 20% (wt/vol) denaturing gel showing the synthesis of the transcript UpGpGpG (indicated by the arrow, where the underlined nucleotides are 32P labelled) from the nifH promoter by P7SEσ54 in the absence and presence of P7. P7 was added to the reaction at different stages, indicated by the numerals I–IV: I = prior to holoenzyme formation, II = prior to RPc formation, III = prior to RPo formation, and IV = after RPo formation. The percentage of synthesised UpGpGpG (%A) indicates the activity of the RNAP in the presence of P7 compared to reactions with no P7 present (lane 1). All data obtained in at least three independent experiments fell within 5% of the relative %A value shown. (c) An autoradiograph of a 20% (wt/vol) denaturing gel showing the synthesis of the transcript ApApUpU (indicated by the arrow, where the underlined nucleotides are 32P labelled) from the lacUV5 promoter by P7SEσ70, where P7 was added to the reaction at different stages, indicated by the numerals I–III: I = prior to holoenzyme formation, II = prior to RPc formation, and III = after RPo formation. The percentage of synthesised ApApUpU (%A) indicates the activity of the RNAP in the presence of P7 compared to reactions with no P7 present (lane 1). In at least three independent experiments, all data obtained fell within 5% of the %A value shown. (d) Autoradiograph of 4.5% (wt/vol) native polyacrylamide gel showing results from an EMSA to determine whether P7 affects the formation of an activation-dependent (here using phage shock protein F1–275 and 4 mM dGTP), remodelled σ54–DNA complex (indicated as super-shifted or ssσ54–32P–nifH). In lanes 5–9, the presence of increasing amounts of P7 had no detectable effect on ssσ54–32P–nifH formation. (a–d) In the schematic above, autoradiograph images indicate the concentration of reaction components, the point in time they were added to the reactions and the incubation times; the migration positions of protein and DNA components in each lane are indicated, and the assays were conducted essentially as previously described. The experiments in (a–d) were conducted as described in Refs [26], [30], [24], respectively.