Integration of regulatory signals through involvement of multiple global regulators:: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR
Integration of regulatory signals through involvement of multiple global regulators:: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR
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DOI：
10.1186/1471-2180-7-2
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发表时间：
2007-01-17
期刊：
影响因子：
4.2
通讯作者：
Matthews, Rowena G.
中科院分区：
文献类型：
--
作者：
Paul, Ligi;Mishra, Pankaj K.;Matthews, Rowena G.
背景：谷氨酸合酶操纵子（gltBDF）是大肠杆菌氨同化的两条主要途径之一。在七个最全球化的调控因子中，它们共同影响了大约一半的E。大肠杆菌基因中，两个先前显示对gltBDF转录施加直接的阳性控制：Lrp和IHF。Lrp的参与在两个方面是不寻常的：第一，它是不敏感的，通常的辅调节亮氨酸，第二，Lrp结合超过150 bp的转录起始点上游。有间接证据表明第三个全球监管机构Crp参与其中。鉴于gltBDF的生理重要性以及了解全局调节信号整合的潜在机会，使用体内和体外方法的组合来研究其他调节蛋白的参与，并确定它们的相对结合位置以及彼此之间和与RNA聚合酶（RNAP）的潜在相互作用。CRP和一个更局部的调节因子ArgR直接控制gltBDF的转录，两者都起负作用。Crp-cAMP结合相对于转录起始点以-65.5为中心的序列。在CRP结合位点的保守核苷酸的突变废除了CRP依赖的抑制。ArgR还结合到gltBDF启动子区，Lrp结合位点的上游，并降低转录。RNAP仅在两种测试条件下产生确定的DNA酶I足迹：在Lrp和IHF两者存在下，或在Crp-cAMP存在下。在Lrp和IHF存在下，RNAP的DNA酶I足迹被ArgR改变。大肠杆菌的gltBDF转录控制的最全球性的调节蛋白是惊人的，但似乎与中央代谢的作用，这个操纵子。确定gltBDF的激活和抑制机制超出了本研究的范围。然而，结果与以下模型一致，其中IHF使DNA弯曲以允许Lrp和RNAP之间的稳定接触，ArgR干扰这种接触，并且Crp在DNA中引入干扰性弯曲和/或使RNAP稳定在平衡但非活性状态。
Background: The glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF. The involvement of Lrp is unusual in two respects: first, it is insensitive to the usual coregulator leucine, and second, Lrp binds more than 150 bp upstream of the transcription starting point. There was indirect evidence for involvement of a third global regulator, Crp. Given the physiological importance of gltBDF, and the potential opportunity to learn about integration of global regulatory signals, a combination of in vivo and in vitro approaches was used to investigate the involvement of additional regulatory proteins, and to determine their relative binding positions and potential interactions with one another and with RNA polymerase (RNAP).Results: Crp and a more local regulator, ArgR, directly control gltBDF transcription, both acting negatively. Crp-cAMP binds a sequence centered at -65.5 relative to the transcript start. Mutation of conserved nucleotides in the Crp binding site abolishes the Crp-dependent repression. ArgR also binds to the gltBDF promoter region, upstream of the Lrp binding sites, and decreases transcription. RNAP only yields a defined DNAse I footprint under two tested conditions: in the presence of both Lrp and IHF, or in the presence of Crp-cAMP. The DNAse I footprint of RNAP in the presence of Lrp and IHF is altered by ArgR.Conclusion: The involvement of nearly half of E. coli's most-global regulatory proteins in the control of gltBDF transcription is striking, but seems consistent with the central metabolic role of this operon. Determining the mechanisms of activation and repression for gltBDF was beyond the scope of this study. However the results are consistent with a model in which IHF bends the DNA to allow stabilizing contacts between Lrp and RNAP, ArgR interferes with such contacts, and Crp introduces an interfering bend in the DNA and/or stabilizes RNAP in a poised but inactive state.