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.
Schröder et al. BMC Genomics 2010 11:12 doi:10.1186/1471-2164-11-12