Visual inspection of the catB gene of Pseudomonas putida revealed a site similar to the catR binding consensus sequence. Beta-gal initially indicated this internal binding site (IBS) acted as a repression site for the catBCA operon. Site-directed mutagenesis was used to investigate key nucleotides; the mutant containing the T-N11-A motif showed increased binding over the wild type, while mutations deviating from the catR consensus reduced binding. Beta-gal and gel shift assays were used to visualize the effects of the mutations against the wild type. DNase I protection identified the precise location of the IBS, and the protection pattern was not altered by the presence of the catR inducer (CCM). Assays of nucleotide fragments containing intact RBS, activation binding site (ABS), and IBS demonstrated a 10-fold increase in catR binding affinity for the IBS, suggesting binding is cooperative.
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|
|GCAGACCCTTGTGGTATTGCGTGTTC||PPS_3181, PPS_3180, PPS_3179,||