INTERACTIONS OF THE SIGNAL TRANSDUCTION PROTEIN GLNK

大肠杆菌通过两个信号转导的双环级联调控氮调节(NTR)基因的转录和谷氨酰胺合成酶(GS)的活性，这两个双环级联介导了氮和碳状态的小分子信号谷氨酰胺和2-酮戊二酸的调控。这两辆自行车共享一个共同的感觉单周期，由PII蛋白和尿酰转移酶/尿苷酰消除酶(Utase/UR)组成。Utase/UR在谷氨酰胺含量较低时催化PII的尿苷基化，在谷氨酰胺含量较高时催化PII~UMP的脱尿苷基化。PII和PII~UMP将这一信号传递给两个受体，它们分别是控制NTR基因转录和GS可逆腺苷化的双功能酶。未修饰的PII的活性受与PII结合的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。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，在氮饥饿的细胞中发挥关键作用。此外，当肺炎克雷伯菌的NIFA和NIFL与nif基因报告基因一起被导入大肠杆菌时，GlnK调节控制固氮酶合成的NifA蛋白的活性。PII缺乏调节NIFA的能力。我们提出了旨在阐明GlnK在氮素调节中的作用的实验。生化实验被用来描述GlnK和PII对PII受体调控的差异。GlnK与其中一种受体--激酶/磷酸酶NRII的相互作用，将通过鉴定在这种相互作用中特定缺陷的GlnK的改变形式，以及寻找这些改变NRII的等位基因特异性抑制物来检验。生理学和遗传学实验是为了更准确地确定GlnK在氮素调节中的作用。最后，提出了将GlnK的功能映射到蛋白质结构上的实验。这些实验应该阐明控制氮同化的信号转导系统的设计，以及PII和GlnK与它们的受体和小分子效应器相互作用的机制。

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.