CHARACTERIZATION OF THE FATTY ACID-RESPONSIVE TRANSCRIPTION FACTOR FADR - BIOCHEMICAL AND GENETIC ANALYSES OF THE NATIVE CONFORMATION AND FUNCTIONAL DOMAINS

In Escherichia coli, fatty acid synthesis and degradation are coordina tely controlled at the level of transcription by FadR. FadR represses transcription of at least eight genes required for fatty acid transpor t and beta-oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encodin g the transcriptional regulator of the ace-BAK operon encoding the gly oxylate shunt enzymes, IclR, FadR dependent DNA binding and transcript ional activation is prevented by long chain fatty acyl-CoA. In the pre sent work, we provide physical and genetic evidence that FadR exists a s a homodimer in solution and in vivo. Native polyacrylamide gel elect rophoresis and glycerol gradient ultracentrifugation of the purified p rotein show that native FadR was a homodimer in solution with an appar ent molecular mass of 53.5 and 57.8 kDa, respectively, Dominant negati ve mutations in fadR were generated by random and site-directed mutage nesis, Each mutation mapped to the amino terminus of the protein (resi dues 1-66) and resulted in a decrease in DNA binding in vitro. In an e ffort to separate domains of FadR required for DNA binding, dimerizati on, and ligand binding, chimeric protein fusions between the DNA bindi ng domain of LexA and different regions of FadR were constructed. One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter su lA-lacZ, and beta-galactosidase activities were derepressed by fatty a cids, suggesting that the fusion protein had determinants both for dim erization and ligand binding, These studies support the conclusion tha t native FadR exists as a stable homo-dimer in solution and that deter minants for DNA binding and acyl-CoA binding are found within the amin o terminus and carboxyl terminus, respectively.