Electrophoretic mobility shift assays and isothermal titration calorimetry assays were done showing that HexR recognizes the Pedd, Pzwf, and Pgap-1 promoters with affinity in the nanomolar range. DNA footprinting assays identified the binding site between +30 and +1 at Pzwf, between +16 and +41 at Pedd, and between −6 and +18 at Pgap-1. Based on DNA sequence alignment of the target sites and isothermal titration calorimetry data, two monomers of HexR bind to a pseudopalindrome with a consensus sequence of 5′-TTGTN7–8ACAA-3′. Binding of the Entner-Doudoroff pathway intermediate 2-keto-3-deoxy-6-phosphogluconate to HexR released the repressor from its target operators, whereas other chemicals such as glucose, glucose 6-phosphate, and 6-phosphogluconate did not induce complex dissociation.
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|