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首页> 外文期刊>Journal of bacteriology >Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli.
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Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli.

机译：大肠杆菌硝酸盐依赖性两组分调节系统的NarQ，NarX和NarL蛋白的磷酸化和去磷酸化。

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The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.
机译：NarX，NarQ和NarL蛋白组成了一个硝酸盐响应性调节系统，负责控制大肠杆菌中的厌氧呼吸途径基因，包括硝酸盐还原酶（narGHJI），二甲基亚砜/三甲胺-N-氧化物还原酶（dmsABC）和富马酸酯还原酶（frdABCD）操纵子等。两种与膜结合的蛋白NarX和NarQ可以独立检测硝酸盐的存在，并将该信号传递至与DNA结合的调节蛋白NarL，后者通过转录激活或抑制来控制基因表达。为了确定蛋白质磷酸化在此过程中的作用，并确定NarX和NarQ蛋白质与NarL的相互作用是否不同，NarX和NarQ的胞质结构域被过量生产和纯化。两种蛋白质在存在[γ-32P] ATP和MgCl2的情况下均被自身磷酸化，但没有与[α-32P] ATP结合。尽管这些自磷酸化反应不受硝酸盐，钼酸盐，GTP或AMP的影响，但ADP是抑制剂。 'NarX和'NarQ的磷酸化形式在室温下稳定数小时。每个蛋白质将其磷酸基转移到纯化的NarL蛋白上，尽管'NarQ-磷酸盐催化转移反应的速度明显快于'NarX-磷酸盐。此外，NarL被乙酰磷酸磷酸化，而不是被ATP磷酸化。 NarL-磷酸盐保持磷酸化至少3小时。但是，添加'NarX会导致NarL-磷酸盐快速去磷酸化。相反，'NarQ与NarL-磷酸盐相比，磷酸酶活性要慢得多。这些研究确定了两个硝酸盐传感器'NarX和'NarQ的胞质结构域在与NarL相互作用的能力上有所不同。

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