INTERACTIONS OF THE SIGNAL TRANSDUCTION PROTEIN GLNK

大肠杆菌通过两个信号转导双环级联来控制氮调节（Ntr）基因的转录和谷氨酰胺合成酶（GS）的活性，这两个信号转导双环级联通过氮和碳状态的小分子信号谷氨酰胺和2-酮戊二酸来介导控制。 这两种自行车共享由PII蛋白和尿苷酰转移酶/尿苷酰去除酶（Utase/UR）组成的共同感觉单环。 UTase/UR在谷氨酰胺低时催化PII的尿苷酰化，在谷氨酰胺高时催化PII~UMP的脱尿苷酰化。 PII和PII~UMP将此信号传递给两个受体，这两个受体是控制Ntr基因转录和GS可逆腺苷酸化的双功能酶。 未修饰的PII的活性受结合PII的2-酮戊二酸调节。 因此，拮抗的碳和氮信号通过不同的机制控制PII的活性。E.大肠杆菌含有第二种PII蛋白GlnK，它只存在于氮饥饿的细胞中。 GlnK通过控制PII受体在氮饥饿细胞中起关键作用。 此外，当来自肺炎克雷伯氏菌的NifA和NifL被引入到大肠杆菌中时，GlnK调节控制固氮酶合成的NifA蛋白的活性。大肠杆菌沿着与nif基因报告基因。 PII缺乏调节NifA的能力。我们提出了旨在阐明GlnK在氮调节中的作用的实验。 生化实验拟描绘GlnK和PII的PII受体的调节的差异。 GlnK与这些受体之一，激酶/磷酸酶NRII的相互作用，将通过表征在这种相互作用中特异性缺陷的GlnK的改变形式，并通过寻找这些改变NRII的等位基因特异性抑制剂来检查。 生理和遗传实验提出更精确地定义GlnK在氮调节中的作用。 最后，实验设计的GlnK映射到蛋白质结构的功能。 这些实验将阐明控制氮同化的信号转导系统的设计，以及PII和GlnK与其受体和小分子效应器的相互作用机制。

Escherichia coli controls the transcription of nitrogen-regulated (Ntr) genes and the activity of the glutamine synthetase (GS) with two signal-transducing bicyclic cascades, that mediate control by the small molecule signals of nitrogen and carbon status, glutamine and 2-ketoglutarate. The two bicycles share a common sensory monocycle consisting of the PII protein and uridylytransferase/uridylyl-removing enzyme (Utase/UR). Utase/UR catalyzes the uridylylation of PII when glutamine is low and deuridylylation of PII~UMP when glutamine is high. PII and PII~UMP transmit this signal to two receptors, which are bifunctional enzymes controlling Ntr gene transcription and the reversible adenylylation of GS. The activity of unmodified PII is regulated by 2-ketoglutarate, which binds PII. Thus, the antagonistic carbon and nitrogen signals control the activity of PII, by different mechanisms. E. coli contains a second PII protein, GlnK, which is only present in nitrogen-starved cells. GlnK plays a key role in nitrogen-starved cells, by controlling the PII receptors. In addition, GlnK regulates the activity of the NifA protein controlling nitrogenase synthesis when NifA and NifL from Klebsiella pneumoniae are introduced into E. coli along with a nif gene reporter. PII lacks the ability to regulate NifA. We propose experiments designed to elucidate the role of GlnK in nitrogen regulation. Biochemical experiments are proposed to delineate the differences in the regulation of the PII receptors by GlnK and PII. The interaction of GlnK with one of these receptors, the kinase/phosphatase NRII, will be examined by characterizing altered forms of GlnK specifically defective in this interaction, and by searching for allele specific suppressors of these that alter NRII. Physiological and genetic experiments are proposed to more precisely define the role of GlnK in nitrogen regulation. Finally, experiments designed to map the functions of GlnK onto the protein structure are proposed. These experiments should elucidate the design of the signal transduction system controlling nitrogen assimilation, and the mechanisms of interaction of PII and GlnK with their receptors and small molecule effectors.