Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A.
The Escherichia coli fadR gene product, FadR, is a multifunctional regulator of fatty acid metabolism. In this work we have purified FadR by a two-step procedure employing two ion-exchange columns. The amino-terminal sequence of the purified protein confirms the sequence of the protein derived from analysis of the DNA sequence (DiRusso, C. C. (1988) Nucleic Acids Res. 16, 7995-8009) and indicates that the initiating methionine is cleaved from the mature protein. Purified FadR binds to a 326-base pair HaeIII fragment of fadB DNA which carries the fadB promoter. DNase I footprinting localizes the operator to a sequence, 5' ATCTGGTACGACCAGAT 3', at +1 to +17 nucleotides relative to the start of transcription. Using protein-DNA gel retention assays, we estimate the Keq of FadR binding to the fadB operator to be approximately 3 x 10(-10) M. Binding of FadR is specifically inhibited by long chain fatty acyl-CoA compounds. The apparent Ki values for oleoyl-CoA, palmitoyl-CoA, and palmitoleoyl-CoA are each 5 nM while that of myristoyl-CoA is 250 nM. Decanoyl-CoA, crotonoyl-CoA, and free fatty acids inhibited binding only at concentrations above 1 microM.
DiRusso
C C
CC
Department of Biochemistry, University of Tennessee, Memphis 38163.
Heimert
T L
TL
Metzger
A K
AK
D000214:Acyl Coenzyme A / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002853:Chromatography, Liquid; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D014158:Transcription, Genetic
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C057119:FadR protein, Bacteria; D005227:Fatty Acids; D012097:Repressor Proteins