FIG. 4.

Analysis of FNR D154A-dependent open complex formation and transcription. (a) In vitro transcription analysis. The FF(−41.5)/pSR, FF(−85.5)FF(−41.5)/pSR, and FF(−90.5)FF(−41.5)/pSR constructs were used as templates for in vitro transcription. Eight nanomolar template plasmid DNA was incubated with 50 nM RNA polymerase, nucleoside triphosphates, and [α-³²P]UTP, with or without FNR D154A (0 to 0.5 μM). Transcripts were analyzed on a denaturing polyacrylamide gel. The control RNA1 and the FNR-dependent transcript are marked. Samples were loaded as follows: no FNR D154A (lanes 1, 5, and 9); 0.05 μM FNR D154A (lanes 2, 6, and 10); 0.1 μM FNR D154A (lanes 3, 7, and 11); 0.5 μM FNR D154A (lanes 4, 8, and 12). (b) Permanganate footprint analysis. Shown is a phosphor image of a denaturing polyacrylamide sequencing gel on which DNA cleavage due to attack by permanganate was analyzed. [γ-³²P]ATP-labeled promoter fragments were incubated with or without FNR D154A (1 μM) and with or without purified RNA polymerase σ⁷⁰ holoenzyme (50 nM) prior to treatment with permanganate. The gel is calibrated with Maxam-Gilbert G+A sequencing reactions. The locations of the permanganate-induced cleavage sites and the FNR consensus binding sites are marked. Samples were loaded as follows: G+A sequencing reaction (M); 1 μM FNR D154A (lanes 1 and 4); 1 μM FNR D154A, 50 nM RNAP (lanes 2 and 5); 50 nM RNAP (lanes 3 and 6).