This proposal describes a two-year project to investigate the properties of DgdR, a newly discovered prokaryotic repressor from Pseudomonas cepacia. DgdR is encoded by the 839-bp dgdR gene, which diverges from and is just ahead of dgdA, the structural gene of a 2,2-dialkylglycine decarboxylase. As we and others have shown by sequence alignment, DgdR is a member of the LysR family of DNA binding proteins. The long range objective of the project is to explain in structural and mechanistic terms why, although they have similar sequences, this protein is a repressor of transcription, while the rest of the LysR proteins but one are activators. We have obtained evidence from gel mobility shift assays using a partially purified repressor preparation and DNA segments including the 5'ends of dgdA and dgdR that (i) the repressor may form a loop between two operators, one 350 nucleotides into the dgdA gene, and the other 200 nucleotides upstream, and (ii) the loop is broken in vitro by 2-methylalanine, an amino acid that induces dgdA expression in vivo. A model is proposed for the dgd system that includes formation of dimer-DNA, tetramer-DNA, and looped tetramer-DNA complexes. We propose to first purify the repressor protein in quantity using recombinant DNA techniques, then repeat our preliminary gel mobility shift experiments using the purified protein. DNase I footprinting with and without various amino acids will provide a direct test of the looping postulate. Also, the shape and surface properties of the repressor's amino acid binding site will be probed using dialkylglycine analogs that differ by the size and hydrophobicity of the alpha substituents. Various polyfluoro dialkylglycines previously synthesized in this lab will be tested in vivo and in vitro as potential inducers, as well as amines and carboxylic acids that mimic other portions of the known dialkylglycine inducers. The proposed research will provide new information about a fundamental genetic control mechanism that is probably widespread in prokaryotes, and thus is likely to be directly involved in many bacterial disease processes.
