Stoichiometry of BkdR to substrate DNA in Pseudomonas putida

BkdR is the transcriptional activator of the bkd operon of Pseudomonas putida, which encodes branched chain keto acid dehydrogenase. BkdR binds to a large region of DNA between its own structural gene and the first gene of the bkd operon. The object of the present studies was to determine the stoichiometry of binding as part of an effort to understand the action of BkdR in regulation of the bkd operon. [S-35]BkdR was prepared and found to be essentially 100% active in the gel shift assay. Only one complex was formed under all the conditions used. The stoichiometry of BkdR binding to its specific substrate DNA was three tetramers per mole substrate DNA. L-valine did not affect the stoichiometry although this ligand was previously shown to affect the DNase I protection pattern. The addition of nonspecific DNA to the incubation mixture also did not affect this stoichiometry. (C) 1996 Academic Press, Inc.