FIG.3.
In vitro metal-dependent binding of NikR and Fur proteins to the PnikR and PexbB promoters. DNase I footprinting with the NikR (A) or Fur (untagged and purified as described in reference 12) (B) proteins on a probe of the nikR-exbB intergenic region and in binding buffer containing different metals (100 μM NiCl2, 100 μM MnCl2) or metal chelators (100 μM EDTA, 100 μM dipyridyl) as indicated. The probe used consists of a 407-bp EcoRI-BamHI fragment containing the nikR-exbB intergenic region labeled at its BamHI site. The 5′-end-labeled probe was incubated with increasing amounts of purified protein: from lanes 1 to 5 corresponding to 0, 0.033, 0.1, 0.3, and 1 μM purified NikR tetrameric protein, respectively, in panel A and from lanes 1 to 5 corresponding to 0, 0.055, 0.165, 0.5, and 1.5 μM purified Fur dimer, respectively, in panel B. The vertical open and filled boxes on the right of each panel indicate the areas of DNase I protection resulting from binding of NikR and Fur proteins, respectively, and the numbers indicate the boundaries of the operators with respect to the +1 transcriptional start site of the PexbB and PnikR respective promoters as indicated. The arrowhead indicates a band of hypersensitivity that appears at high concentrations of rNikR protein on the PnikR promoter at approximately −6 with respect to transcriptional +1. The G+A lane is a G+A sequence reaction on the DNA probe used as size marker (19). The bent arrows to the left of the panels show the direction of transcription and position of the +1 of the PexbB and PnikR respective promoters within the probe. The converging arrows mark the position of a previously proposed NikR binding site (7, 9).