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 As

C. C. DiRusso, T. L. Heimert, A. K. Metzger

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Abstract

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 K(eq) 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 K(i) 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 μM.

Original languageEnglish (US)
Pages (from-to)8685-8691
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number12
StatePublished - Jan 1 1992

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Coenzymes
Metabolism
Escherichia coli
Acyl Coenzyme A
Fatty Acids
Coenzyme A
Palmitoyl Coenzyme A
Proteins
Ion Exchange
Deoxyribonuclease I
DNA
Protein Sequence Analysis
Nonesterified Fatty Acids
Base Pairing
Methionine
Nucleic Acids
DNA sequences
Transcription
Nucleotides
Gels

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "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 K(eq) 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 K(i) 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 μM.",
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T1 - 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 As

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AU - Heimert, T. L.

AU - Metzger, A. K.

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N2 - 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 K(eq) 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 K(i) 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 μM.

AB - 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 K(eq) 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 K(i) 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 μM.

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