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Volume 272, Number 49, Issue of December 5, 1997 pp. 30645-30650
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the Fatty Acid-responsive Transcription Factor FadR
BIOCHEMICAL AND GENETIC ANALYSES OF THE NATIVE CONFORMATION AND FUNCTIONAL DOMAINS

(Received for publication, August 13, 1997, and in revised form, September 17, 1997)

Narayan Raman , Paul N. Black ^§ and Concetta C. DiRusso ^§

From the  Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 and the ^§ Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208

In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the level of transcription by FadR. FadR represses transcription of at least eight genes required for fatty acid transport and -oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encoding the transcriptional regulator of the aceBAK operon encoding the glyoxylate shunt enzymes, IclR. FadR-dependent DNA binding and transcriptional activation is prevented by long chain fatty acyl-CoA. In the present work, we provide physical and genetic evidence that FadR exists as a homodimer in solution and in vivo. Native polyacrylamide gel electrophoresis and glycerol gradient ultracentrifugation of the purified protein show that native FadR was a homodimer in solution with an apparent molecular mass of 53.5 and 57.8 kDa, respectively. Dominant negative mutations in fadR were generated by random and site-directed mutagenesis. Each mutation mapped to the amino terminus of the protein (residues 1-66) and resulted in a decrease in DNA binding in vitro. In an effort to separate domains of FadR required for DNA binding, dimerization, and ligand binding, chimeric protein fusions between the DNA binding domain of LexA and different regions of FadR were constructed. One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter sulA-lacZ, and -galactosidase activities were derepressed by fatty acids, suggesting that the fusion protein had determinants both for dimerization and ligand binding. These studies support the conclusion that native FadR exists as a stable homo-dimer in solution and that determinants for DNA binding and acyl-CoA binding are found within the amino terminus and carboxyl terminus, respectively.

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