Figure 8

Agarose gels of DNA band shift assays with purified Zur protein. (A), The DNA band shift assays with fluorescein-labeled 40-mers covering the candidate Zur-binding sites in the cg0042-cg0043 intergenic region and in front of cg0794, cg0795, cg2911, and cg3107. DNA band shift assays were performed with 40 pmol of streptavidin-tagged Zur protein incubated with 0.05 pmol of fluorescein-labeled, double-stranded 40-mer DNA fragments. The assays were performed in the absence of zinc ions and in the presence of 50 μM ZnCl₂. Lanes 1: control assays without Zur protein; lanes 2: DNA band shift assays with added Zur protein. The negative control assay was performed with a 40-mer deduced from the upstream region of cg0841. (B), DNA band shift assays with mutated versions of the 40-mers. Mutated versions were generated by introducing transitions into the candidate Zur-binding sites or into the genomic flanking regions. The EMSAs were carried out in the presence of 50 μM ZnCl₂. (C), DNA band shift assays with binding buffers containing varying metal ions. The EMSAs were performed in the presence of 50 μM ZnCl₂, MgSO₄, NiCl₂, CuSO₄, MnSO₄, or FeSO₄ with the 40-mer region representing cg2911.