DNAse I footprinting in a previous study identified a putative NikR binding site. EMSA and a second DNAse I footprinting confirmed specific, Ni2+ dependent binding. Dimethylsulfate protection footprinting identified two sites of close contact between the DNA and NikR, termed half-sites, separated by 16 base pairs. Symmetric double mutants were created for each position in the half-sites, along with a single 11-base mutant in the central region. EMSA illustrated affinity differences between mutants, which clarified the precise binding site. Beta-gal assay of the nikABCDE operon promoter in the wild-type demonstrated in vivo repression by NikR.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|
|GTATGACGAATACTTAAAATCGTCATAC||nikD, nikC, nikB, nikA, nikE,||