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.
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.
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DOI：
10.1016/s0021-9258(18)42497-0
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发表时间：
1992-04
期刊：
影响因子：
--
通讯作者：
C. DiRusso;Tamra L. Heimert;A. Metzger
中科院分区：
文献类型：
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作者：
C. DiRusso;Tamra L. Heimert;A. Metzger
大肠杆菌fadR基因产物fadR是脂肪酸代谢的多功能调节剂。在这项工作中，我们用两个离子交换柱的两步法纯化了FadR。纯化蛋白的氨基末端序列与DNA序列分析得到的蛋白序列一致（DiRusso， C. C. (1988) Nucleic Acids Res. 16, 7995-8009），表明起始蛋氨酸是从成熟蛋白中切割出来的。纯化的FadR与携带fadB启动子的326碱基对HaeIII片段结合。DNase I足迹将操作符定位到一个序列，5 ' ATCTGGTACGACCAGAT 3 '，相对于转录起始位置+1到+17个核苷酸。使用蛋白质- dna凝胶保留测定，我们估计FadR与fadB操作符结合的Keq约为3 × 10(-10) M. FadR的结合被长链脂肪酸酰基辅酶a化合物特异性抑制。油酰辅酶a、棕榈酰辅酶a和棕榈酰辅酶a的表观Ki值均为5 nM，肉豆蔻酰辅酶a的表观Ki值为250 nM。十烷酰辅酶a、巴豆酰辅酶a和游离脂肪酸仅在浓度大于1微米时抑制结合。
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.