Figure 4: Footprinting analysis of Zur binding to the zitB promoter region.

(a) Determination of the zitB TSS by high-resolution S1 mapping. Exponentially grown cells were treated with ZnSO₄ at 100 μM for 1 h before RNA preparation. The 5′ end position of the zitB transcript (+1) was determined by electrophoresis of S1-protected DNAs on a 6% polyacrylamide gel containing 7 M urea, along with sequencing ladders generated with the SCC5F2A cosmid DNA and the same primer used to generate the S1 probe. The position of the longest protection was assigned as +1. (b) The positions of the predicted −10 and −35 elements of the zitB promoter and the Zur-box motif. (c,d) DNase I footprinting patterns of Zur-zitB DNA interaction as analysed by capillary electrophoresis. (c) Footprinting under varying Zur protein concentrations. The DNA probe containing the zitB gene from +39 to −228 nt relative to the TSS (+1) was incubated with increasing amounts of Zur (0.45, 0.9, 1.8 and 9.0 μM) in the presence of 75 μM ZnSO₄. The DNA probe only with no added Zur nor zinc was analysed in parallel. The primary protection from −40 to −78, and the extended footprint at higher Zur up to −138 were indicated with dotted lines. (d) Footprinting under varying zinc concentrations. DNase I footprinting analysis was done with the same DNA probe, but with fixed amount of Zur at 2.7 μM, and increasing amount of ZnSO₄ (2.5, 5.0, 7.5 and 10.0 μM) in each binding reaction. Zinc-dependent extension of Zur footprint on the zitB promoter was shown.