The well-defined HpNikR target urease operator OPureA was selected to investigate the effects of alternative cations on HpNikR-DNA binding. EMSA demonstrated strong binding in the presence of Ni2+ without secondary cations, demonstrating Ni2+ to be sufficient for HpNikR-OPureA binding. DNAse I footprinting further determined Ni2+ to be necessary for binding in the absence of other divalent cations. Parallel footprinting with Zn2+, Co2+, Mg2+, and Ca2+ showed they were capable of promoting HpNikR-DNA binding only to a lesser extent. Isothermal titration calorimetry indicated Mg2+ and Ca2+ effected binding only through “a nonspecific cation effect,” and only in great concentrations. Zn2+ promoted binding most strongly (after Ni2+), with Co2+ interacting with HpNikR in a manner analogous to Zn2+ but to greatly reduced effect. Fluorescence spectroscopy indicated Ni2+, not Zn2+, selectively drives “a conformational rearrangement of HpNikR.” Repeated EMSAs and ITC experiments confirmed Ni2+ alone induces high affinity DNA binding by HpNikR.
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|