DNA-binding activities of Rv2034 wild type and mutant proteins.

<p>(<b>A</b>) The effects of Rv2034 and its mutant variants on gene expression were measured by β-galactosidase activity assays. The names of <i>lacZ</i>-fused plasmids are listed in the left panel, and the elements differing among the plasmids are shown in the middle panel. β-galactosidase activities are presented as Miller units in the right panel. The values reported are the averages of three independent experiments. Error bars represent standard deviations. (<b>B</b>) Diagram illustrating Rv2034 variants tested in our assays. The dimerization interface and putative zinc binding sites are illustrated. The Rv2034AE (A32G, E35G) and Rv2034C61A variants were produced by site-directed mutagenesis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036255#s4" target="_blank">Materials and Methods</a>. The Rv2034ΔN28 variant was produced by deleting the first 28 amino acid residues from the N-terminal of Rv2034. (<b>C</b>) EMSA assays for the binding of Rv2034 variants to the Rv2034p promoter DNA. The mutant variants of Rv2034 were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036255#s4" target="_blank">Materials and Methods</a> and co-incubated with the DNA substrate labeled with [γ- ³²P]. The final concentrations of the proteins were 0, 0.1, 0.2, and 0.4 µM in lanes 1, 2, 3 and 4, respectively.