Earlier studies had identified a putative palindrome binding motif for HpNikR. A Fluorescence Anisotropy assay was developed to study HpNikR binding to DNA; competitive assays with a non-fluorescent probe confirmed the fluorescein molecule did not interfere with DNA binding. EMSAs confirmed the results of the FA assays. Competitive FA assays were also performed with an oligonucleotide probe lacking the palindrome sequence (replaced with cytosine bases), which confirmed that the putative palindrome is required for HpNikR binding. Further, FA studies revealed a required coordination of a second, specific low-affinity metal ion in addition to the high-affinity metal ion. Testing demonstrated Mg(II), Ca(II), and Mn(II) facilitated binding in this way, while Zn(II), Co(II), and Ni(II) did not.
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|