Figure 3

Agarose gels of DNA band shift assays performed with purified His-tagged DtxR protein. (A) DNA band shift assays with a Cy3-labeled double-stranded 40 mer covering the predicted DtxR binding sites in front of the cg0771 gene. Band shifts were performed with 0.05 pmol of the labeled 40 mer DNA fragment and different amounts of the His-tagged DtxR protein. Assays were separated in a 2% agarose gel and visualized by fluorescence imaging. Lane 1: control assay without DtxR protein; lane 2: control assay without 40 mer; lane 3: band shift assay with 42 pmol DtxR; lane 4: assay with 84 pmol DtxR; lane 5: assay with 126 pmol DtxR; lane 6: assay with 168 pmol DtxR; lane 7: assay with 210 pmol DtxR. (B) Control experiments with Cy3-labeled 40 mers deduced from internal gene regions of cg0397 and cg0738 (dnaE2). Lanes 1: control assay without DtxR protein; lanes 2: control assay containing 42 pmol DtxR protein; lanes 3: assays with 84 pmol DtxR; lanes 4: assays with 126 pmol DtxR. (C) DNA displacement experiments with a Cy3-labeled double-stranded 40 mer covering the predicted DtxR binding sites in front of the cg0771 gene. During displacement studies, 42 pmol of purified His-tagged DtxR protein and 0.05 pmol of a Cy3-labeled 40 mer along with increasing concentrations of the same non-labeled 40 mer fragment were added to assay. Lane 1: control assay without purified DtxR protein; lane 2: control assay without non-labeled 40 mer; lane 3: assay with 0.015 pmol non-labeled 40 mer; lane 4: assay with 0.3 pmol non-labeled 40 mer; lane 5: assay with 0.45 pmol non-labeled 40 mer; lane 6: assay with 1 pmol non-labeled 40 mer. (D) Verification of the predicted DtxR binding sites by DNA band shift assays using 0.05 pmol of Cy3-labeled 40 mers and 42 pmol of purified His-tagged DtxR protein. Gene identifiers are shown below the agarose gels. Lanes 1: control assay without DtxR protein; lanes 2: DNA band shift assay containing DtxR protein.