INRA, UMR1319 Micalis, 78350 Jouy-en-Josas, France, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud 11, 91405 Orsay, France, Proteome Center Tübingen, University of Tübingen, 72076 Tübingen, Germany and Department of Chemical and Biological Engineering, Chalmers University of Technology, 41296 Gothenburg, Sweden.
Nucleic Acids Res. 2013 Nov;41(20):9371-81. doi: 10.1093/nar/gkt709. Epub 2013 Aug 11.
Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.
细菌拥有专门用于抑制细胞色素 P450 基因的转录调节剂（属于 TetR 家族），这些基因参与多不饱和脂肪酸的氧化。这些抑制剂与操纵序列的相互作用在脂肪酸存在的情况下被破坏，因此它们被称为脂肪酸置换调节剂。在这里，我们描述了一种新的失活这些蛋白质与 DNA 相互作用的机制，以枯草芽孢杆菌调节剂 FatR 为例进行说明。FatR 在双杂交测定中被发现与 TkmA 相互作用，TkmA 是蛋白酪氨酸激酶 PtkA 的激活剂。我们表明，FatR 在其螺旋-转角-螺旋结构域中的酪氨酸 45 残基上被 PtkA 特异性磷酸化。结构建模表明，酪氨酸 45 的羟基与 DNA 相互作用，我们表明这种磷酸化降低了 FatR 的 DNA 结合能力。模拟体内 FatR 磷酸化的点突变导致 fatR 操纵子的强烈去抑制，表明这种调控机制独立于多不饱和脂肪酸的去抑制作用。酪氨酸 45 是一个高度保守的残基，枯草芽孢杆菌的 PtkA 可以磷酸化来自其他细菌的 FatR 同源物。这表明酪氨酸 45 的磷酸化可能是细菌脂肪酸置换调节剂失活的一种普遍机制。
