Eight putative binding sites for HpNikR were drawn from previous studies: ureA, nixA, frpB4, fecA3, exbB, nikR, and fur-OPI & II. Dissociation constants for each sequence were determined by competitive fluorescence anisotropy, and could be divided into “weak-binding” and “tight-binding” groups. The sites ureA, nixA, frpB4, and fecA were tight-binding, with dissociation constants ranging from 32 to 68 nM, while exbB, nikR, and fur-OPI & II were weak-binding, with dissociation constants between 1050 and 11800 nM. EMSA confirmed these trends, and sequence alignment was used to determine a binding motif from all eight putative sights. Additional fluorescence anisotropy assays were performed on altered ureA sites: mutagenesis of either palindrome half-site to all cytosines resulted in dramatic loss of affinity, while mutation of the first half-site into a perfect palindrome of the second resulted in reduced affinity (though still classifiable as tight-binding). Mutation of the AT rich region surrounding the palindromes had no effect on the dissociation constant.
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|