FIG. 2.

DNase I footprinting analysis of wild-type and F98A CodY binding to the ilvB promoter in the presence and absence of effectors. CodY proteins were incubated with an end-labeled DNA fragment that includes the previously described high-affinity binding site II within the ilvB promoter region (44) and then challenged with DNase I before electrophoresis under denaturing conditions (see Materials and Methods for details). (A) Increasing concentrations of wild-type (WT) and F98A CodY proteins were incubated with the ilvB probe in the absence (−) or presence (+) of 4 mM GTP as indicated above the gel. (B) Mutant and wild-type (WT) CodY proteins (125 nM) were incubated with the ilvB probe in the presence of increasing concentrations of l-isoleucine as indicated above the gel (−, no l-isoleucine). (C) Increasing concentrations of mutant and wild-type CodY proteins were incubated with the ilvB probe in the presence of 10 mM each of isoleucine, leucine, and valine. The concentrations (in nanomolar) of wild-type CodY protein (lanes 1 to 6) and F98A CodY protein (lanes 7 to 14) in the various lanes were as follows: 0 (lane 1), 6 (lane 2), 12 (lane 3), 25 (lane 4), 50 (lane 5), 100 (lane), 6 (lane 7), 12 (lane 8), 25 (lane 9), 50 (lane 10), 100 (lane 11) 200 (lane 12), 400 (lane 13), and 800 (lane 14). (D) Increasing concentrations of F98A mutant CodY protein was incubated with the ilvB probe in the presence or absence of 10 mM each of isoleucine, valine, and leucine (ILV) as indicated above the gel. The concentration (in nanomolar) of CodY in the various lanes were as follows: 0 (lane 1), 100 (lane 2), 200 (lane 3), 400 (lane 4), 800 (lane 5), 1,600 (lane 6), 0 (lane 7), 6 (lane 8), 12 (lane 9), 25 (lane 10), 50 (lane 11), 100 (lane 12), 200 (lane 13), 400 (lane 14), 800 (lane 15), and 1,600 (lane 16).