FIG. 7.

Damaging P_R obviates RNA polymerase inhibition of repressor-activated P_RM transcription (A and B) or open complex formation (C and D). For panels A and B, DNA fragments containing wild-type P_RM and −10 mutant P_R (see Fig. 1 for sequence) were transcribed in vitro in the absence of repressor (lanes 1) and at various increasing repressor concentrations. Repressor concentrations were increased in 2.5-fold steps starting at 250 nM protein (lanes 2 to 6). Positions of transcripts resulting from initiation of transcription from P_RM and P_R are indicated. (A) The 434 repressor was incubated with DNA template at 23°C for 10 min, followed by addition of RNA polymerase. The reaction mixture was transferred to 37°C for 10 min before the transcription reaction was initiated by the addition of nucleotides and heparin. (B) RNA polymerase was incubated with DNA at 37°C for 10 min before addition of the 434 repressor. After an additional 10-min incubation at 37°C, the transcription reaction was initiated by the addition of nucleotides and heparin. (C and D) Shown are the open complexes formed at P_RM in the absence of the repressor (lanes 1) and the presence of increasing concentrations of the 434 repressor as detected by KMnO₄ footprinting. Repressor concentrations were increased in 2.5-fold steps starting at 250 nM protein. Footprinting conditions are given in Materials and Methods. Positions of P_R and P_RM open complex are indicated. (C) Incubation conditions were as described for panel A. (D) Incubation conditions were as described for panel B. RNAP, RNA polymerase.