Figure 2.

The mutant RNAP promoter complexes are stable at low temperatures. (A) DNase I footprinting and KMnO₄ probing of promoter complexes at different temperatures (bottom strand). Promoter complexes were formed at the indicated temperatures and followed by DNase I and KMnO₄ treatment. (B) KMnO₄ probing of promoter complexes formed at −20°C in 50% glycerol to prevent freezing. The 37°C controls were conducted in the presence (lanes 4 and 6) or the absence (lanes 3 and 5) of 50% glycerol in the reaction buffer. The results indicate that open complex formation and KMnO₄ sensitivity were not affected by the glycerol. See Materials and Methods for experimental details. (C) Quantitative KMnO₄ probing of promoter complexes formed at different temperatures. Promoter complex formation reactions were set up on ice or 37°C, brought to the assay temperature, incubated for 15 min, and probed with KMnO₄. The reaction products were separated by denaturing PAGE (Top) and quantified as described in Materials and Methods (Bottom). KMnO₄ probing was performed at 0 (lanes 1, 12, 13, and 24); 5 (lanes 2, 11, 14, and 23); 10 (lanes 3, 10, 15, and 22); 15 (lanes 4, 9, 16, and 21); 23 (lanes 5, 8, 17, and 20); and 37°C (lanes 6, 7, 18, and 19). The upward vertical arrows indicate an up-temperature shift from 0°C to the assay temperature; downward arrows indicate a down-temperature shift from 37°C to the assay temperature.