FIGURE 4.

In vitro transcription run-off assays showing selective recruitment of different promoters of the rpoE gene with various forms of RNA polymerase. A, RpoS (σ³⁸) and RpoD complexed with the RNAP core initiate transcription from −218 TSS and TSS at −327, respectively. Lane 1 corresponds to the size standard. For lanes 2–4, a DNA template of 105 bp was used. Lane 2 shows the incubation reaction with Eσ⁷⁰, lane 3 the incubation with Eσ³⁸ resulting in the synthesis of expected 45-nt product marked with the arrow. Lane 4 was incubated with Eσ⁷⁰ in the presence of phosphorylated RcsB. Lanes 5 and 6 show RNA transcripts synthesized from DNA template of 170 bp. An expected size of 85-nt RNA product was observed when Eσ⁷⁰ was incubated with the wild-type DNA template (lane 5), whereas only a weak signal was visible when Eσ⁷⁰ was used with template with mutation at −7T(C) and −11A(G) in the −10 element (lane 6). Bands marked with an asterisk (*) symbol in lanes 3 and 5 indicate nonspecific end-to-end transcription reaction products. B, RpoN (σ⁵⁴) complexed with the core RNAP in the presence of either NtrC or QseF can initiate transcription from the rpoEP2 promoter using the −327 TSS. Lane 1 corresponds to size standard, lanes 2 and 4 corresponds to reactions with the wild-type DNA template in the presence of either NtrC or QseF. Lanes 3 and 5 correspond to the RNA transcript produced with DNA template containing mutations at −12 (GC to AT) and −24 (GG to GA).