CatR regulation of the pheBA operon in Pseudomonas putida was investigated by site-directed mutagenesis of the recognition binding site (RBS), activation binding site (ABS), and separating hinge region, identified by DNase I protection in an earlier publication. Beta-gal assay measured the effects of mutations on pheBA expression and EMSA determined effects on binding efficiency. Mutation of the RBS and ABS reduced expression and binding, while increase in hinge-region flexibility increased binding, though only slightly increased expression. RBS and ABS binding was demonstrated to be cooperative (one catR dimer binding to each and forming a tetramer under activating conditions), and mutations in the interrupted inverted repeat in the RBS drastically reduced binding and expression. ABS was shown to be bound only in the presence of the inducer molecule.
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|