Figure 4
From: Identification of ColR binding consensus and prediction of regulon of ColRS two-component system
ColR-protected regions in the promoters of PP0737, PP0035/0036, PP2560/2561, PP0900/0901 and PP3766. (a) DNase I footprinting assay performed on promoter DNAs of PP0737, PP0035/0036, PP2560/2561, PP0900/0901 and PP3766. Lane 1 is always a DNase I-only ladder, following lanes represent different concentrations of DNA-binding protein ColR in a non-phosphorylated (lanes 2 and 3; in case of PP2560/2561 lanes 2 to 4) and phosphorylated state (lanes 4 to 6; in case of PP2560/2561 lanes 5 to 7). Concentrations of ColR in reactions with ColS and ATP were 0.6 μM, 3 μM and 15 μM if DNA upstream of PP3766, PP0900/0901 or PP2560/2561 was used as a binding target, but 0.5 μM, 2.5 μM and 12.5 μM in case of PP0737 and PP0035/0036 promoter DNA. Concentration of ColR-phosphorylating ColS was always 1 μM and ATP was 0.1 mM. Only the two higher concentrations of ColR are used in the binding reactions with non-phosphorylated ColR (lanes 2 and 3), except for PP2560/2561 where all three ColR concentrations are presented (lanes 2 to 4). (b) Sequences of the ColR-protected regions in the promoters of PP0737, PP0035/0036, PP2560/2561, PP0900/0901 and PP3766. Distance from the start codon is shown and the sites hypersensitive to DNase I cleavage in the presence of ColR are highlighted in bold.