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首页> 外文期刊>Journal of Molecular Biology >NITRATE AND NITRITE REGULATION OF THE FNR-DEPENDENT AEG-46.5 PROMOTER OF ESCHERICHIA COLI K-12 IS MEDIATED BY COMPETITION BETWEEN HOMOLOGOUS RESPONSE REGULATORS (NARL AND NARP) FOR A COMMON DNA-BINDING SITE
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NITRATE AND NITRITE REGULATION OF THE FNR-DEPENDENT AEG-46.5 PROMOTER OF ESCHERICHIA COLI K-12 IS MEDIATED BY COMPETITION BETWEEN HOMOLOGOUS RESPONSE REGULATORS (NARL AND NARP) FOR A COMMON DNA-BINDING SITE

机译：大肠埃希菌的FNR依赖性AEG-46.5启动子的硝酸盐和亚硝酸盐调节是通过共同DNA结合位点的同源反应调节剂（NARL和NARP）之间的竞争而介导的。

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The NarL and NarP proteins are homologous response regulators that function to regulate anaerobic respiratory gene expression in response to nitrate and nitrite in Escherichia coli. Expression of the aeg-46.5 operon (anaerobically expressed gene at 46.5 minutes on the genetic map) is induced during anaerobic growth by the global transcriptional regulatory protein Fnr. aeg-46.5 operon expression is further induced by the NarP protein in response to nitrate or nitrite and this induction is antagonized by NarL. We used in vivo and in vitro techniques to investigate how these three transcriptional regulatory proteins control the activity of a single promoter. Deletion and mutational analysis of the aeg-46.5 operon control region identified two distinct cis-acting elements. A sequence with similarity to the Fnr-binding site consensus, centered at position -64.5, was essential for Fnr-dependent anaerobic induction of aeg-46.5 operon expression. In all other naturally occuring Fnr-dependent promoters the primary Fnr-binding site is centered between -40 and -50. The second cis-acting element, a region of perfect symmetry centered at -44.5, shares sequence similarity with the NarL-binding site consensus. This region was required for nitrate and nitrite induction of aeg-46.5 operon expression. We purified the NarP and NarL proteins as maltose-binding protein (MBP) fusion proteins and investigated their interaction with the aeg-46.5 operon control region. Incubation with the phospho-donor, acetyl phosphate, allowed both MBP-NarP and MBP-NarL to protect the -44.5 region of the aeg-46.5 operon control region from DNase I cleavage. Single and double nucleotide substitutions in the -44.5 region reduced or abolished nitrate and nitrite induction of aeg-46.5 operon expression in vivo and prevented the binding of MBP-NarP and MBP-NarL to the control region in vitro. Presumably, the NarP and NarL proteins compete for the -44.5 binding site to regulate aeg-46.5 operon expression in response to nitrate and nitrite. Apparently, only the NarP protein is competent to activate transcription of the aeg-46.5 operon when bound to the -44.5 region. [References: 52]
机译：NarL和NarP蛋白是同源响应调节剂，可响应大肠杆菌中的硝酸盐和亚硝酸盐来调节厌氧呼吸基因的表达。通过整体转录调节蛋白Fnr在厌氧生长过程中诱导aeg-46.5操纵子的表达（在遗传图谱上46.5分钟时厌氧表达的基因）。响应硝酸盐或亚硝酸盐的NarP蛋白进一步诱导aeg-46.5操纵子表达，并且该诱导被NarL拮抗。我们使用了体内和体外技术来研究这三种转录调节蛋白如何控制单个启动子的活性。 Aeg-46.5操纵子控制区的删除和突变分析确定了两个不同的顺式作用元件。以Fnr依赖性厌氧诱导aeg-46.5操纵子表达，以与Fnr结合位点共有序列相似的序列为中心，该序列的中心为-64.5。在所有其他自然存在的Fnr依赖性启动子中，主要的Fnr结合位点位于-40和-50之间。第二个顺式作用元件，一个以-44.5为中心的完全对称区域，与NarL结合位点共有序列具有序列相似性。该区域是硝酸盐和亚硝酸盐诱导aeg-46.5操纵子表达所必需的。我们纯化了作为麦芽糖结合蛋白（MBP）融合蛋白的NarP和NarL蛋白，并研究了它们与aeg-46.5操纵子控制区的相互作用。与磷酸供体乙酰磷酸一起孵育，可使MBP-NarP和MBP-NarL都保护aeg-46.5操纵子控制区的-44.5区免受DNase I裂解。 -44.5区中的单核苷酸和双核苷酸取代在体内减少或消除了aeg-46.5操纵子表达的硝酸盐和亚硝酸盐诱导，并在体外阻止了MBP-NarP和MBP-NarL与对照区的结合。据推测，NarP和NarL蛋白竞争-44.5结合位点，以响应硝酸盐和亚硝酸盐调节aeg-46.5操纵子的表达。显然，当与-44.5区结合时，只有NarP蛋白能够激活aeg-46.5操纵子的转录。 [参考：52]

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