FIG. 5.
Identification of the NikR operator in the PureA promoter. (A) In vitro binding of rNikR to the urease promoter by DNase I footprinting analysis. The probe used consists of a 308-bp EcoRI-BamHI fragment containing the wild-type urease promoter region from pGemUre labeled at its BamHI site. The 5′-end-labeled probe was incubated with an increasing amount of purified NikR protein from lanes 1 to 6 corresponding to 0, 0.004, 0.011, 0.033, 0.10, and 0.30 μM concentrations of protein, respectively. The vertical open box on the right indicates the areas of DNase I protection resulting from binding of NikR; the numbers indicate the boundaries of the operator with respect to the +1 transcriptional start site of the PureA. The G+A lane is a G+A sequence reaction on the DNA probe used as size marker (19). The bent arrow and boxes to the left of the panel show the direction of transcription and position of the +1 and promoter elements of PureA. (B) Conservation and features of urease promoter sequence. Alignment of nucleotide sequences of the urease promoter region from the indicated five different strains of H. pylori (1, 18, 28, 34). The alignment was generated by CLUSTAL W, and the nonconserved nucleotides are highlighted in gray. The transcriptional start site (26) is shown by bent arrows, along with the “extended” −10 and −35 hexamers inferred, which are boxed. The proposed stem-loop structure that consists as the cis-acting NikR-mediated nickel-induction site (34) is indicated in underlined boldface. The NikR-operator as defined by DNase I footprinting in the present study is boxed. (C) Dissection of the NikR operator of the urease promoter. Scanning mutagenesis of the urease operator to determine nucleotides important for binding of NikR. The sequence changes and the fold decreases in affinity compared to the wild type of 10 ccc trinucleotide substitution (underlined in boldface) mutants of the urease operator are shown. By in vitro DNase I footprinting analysis with the purified NikR protein, the relative half-maximal binding was calculated for each mutant operator as the concentration at which 50% of the probe was estimated to be protected, and the relative fold changes in affinity were calculated accordingly. The wild-type operator sequence is shown, and the nucleotides that were determined important for binding are highlighted in gray and black.