Fig 1: leuO expression is regulated by DNA bending. (a) Genetic map showing the upstream region of leuO. The binding region for ToxR, the five binding sites for LeuO (labeled 1–5), and the - 10 and - 35 promoter regions are indicated. The transcription start site (+1) also is indicated. Boxes indicate the location of the deletions. (b) Expression of lacZ fused to wild-type or mutagenized regions upstream of leuO in the presence and absence of exogenous ToxRS in wild type E. coli strain DH5a. The error bars denote standard deviations of the results of three independent experiments (*P < 0.05; NS, not significant). (c) In vitro pull-down assay. Wild-type or mutagenized upstream regions of leuO were incubated with RNA polymerase holoenzyme (New England Biolabs, Beverly, MA) in the presence or absence of ToxR-N, and the reactions were applied to Streptavidin Magnetic Beads (New England Biolabs, Beverly, MA). The resins were washed, and then treated with protein loading buffer as described in Materials and Methods. The precipitates were resolved by SDS-PAGE, and the amount of RNA polymerase was detected by western hybridization using an antibody against the a subunit. (d) In vitro pull-down assay. The wild type region upstream of leuO was incubated with RNA polymerase holoenzyme and ToxR-N with gradually increasing amounts of LeuO. Reactions were then applied to Streptavidin Magnetic Beads, washed, and treated with protein loading buffer as described in Materials and Methods. The precipitates were resolved by SDS-PAGE, and the amount of RNA polymerase was detected by western hybridization using an antibody against the a subunit. (e) Gel-mobility shift assay of a labeled wild-type or mutagenized upstream regions of the leuO promoter region using purified ToxR-N. Lanes 1, 3, 5, 7: no protein. Lanes 2, 4, 6, 8: 100 nM of ToxR-N.