FIG. 4.
Binding of the NikR and Fur proteins at the overlapping and flanking operators of the PnikR or PexbB target promoters, respectively. (A) Summary of the key regulatory elements of the nikR-exbB locus that have been localized in the present study, including a schematic representation of the intergenic region and underneath the double-stranded nucleotide sequence spanning from the ATG translational starts (in boldface) of nikR-exbB genes, with determinants highlighted. The bent arrows indicate the direction of transcription and position of the +1 of the PexbB and PnikR promoters, and the −10 and −35 promoter elements are highlighted in black, respectively. The open boxes represent NikR binding sites or protected nucleotides resulting from the DNase I footprinting assays, and the triangles mark the boundaries of the NikR operators. The hatched gray boxes represent the Fur binding sites or protected nucleotides resulting from the DNase I footprinting assays, and the asterisks mark the boundaries of the Fur operators. The converging arrows highlight the positioning of the palindromic sequence (underlined) that has been proposed until now as the NikR operator within this region (7, 9). (B) Competition or independent binding of NikR and Fur at the operators at the overlapping and flanking operators of the PnikR or PexbB target promoters, respectively. All footprinting assays were performed with the addition of NiCl2 to the binding buffer. An equivalent amount of 5′-end-labeled probe was incubated without any protein (−); with increasing amounts of NikR (panel I) and Fur (panel II), independently; or with fixed relative maximal binding (RMB) concentration of Fur and increasing amounts of NikR (panel III); or with a fixed RMB concentration of NikR and increasing amounts of Fur (panel IV) as indicated above each lane. The RMB concentration for each protein is the minimal concentration tested that allows complete binding at both operators and was determined for each protein independently from panels I and II. The triangles and asterisks indicate the boundaries of the NikR and Fur operators, respectively. The arrowhead indicates a band of hypersensitivity that appears at high concentrations of NikR protein on the PnikR promoter.