INTERACTIONS OF THE SIGNAL TRANSDUCTION PROTEIN GLNK

大肠杆菌通过两个信号转导双环级联控制氮调节基因（Ntr）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、谷氨酰胺和2-酮戊二酸盐的小分子信号介导控制。这两个环共享一个由PII蛋白和尿苷转移酶/尿苷去除酶（Utase/UR）组成的共同感觉单环。Utase/UR在谷氨酰胺含量低时催化PII尿苷化，在谷氨酰胺含量高时催化PII~UMP去尿苷化。PII和PII~UMP将该信号传递给两个受体，这两个受体是控制Ntr基因转录和GS可逆腺苷化的双功能酶。未修饰的PII的活性受2-酮戊二酸调节，它与PII结合。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，这种蛋白只存在于缺氮细胞中。GlnK通过控制PII受体在缺氮细胞中起关键作用。此外，当来自肺炎克雷伯菌的NifA和NifL与nif基因报告基因一起引入大肠杆菌时，GlnK调节控制氮酶合成的NifA蛋白的活性。PII缺乏调控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。我们建议用生化实验来描述GlnK和PII对PII受体调控的差异。GlnK与这些受体之一的激酶/磷酸酶NRII的相互作用，将通过表征GlnK在这种相互作用中特异性缺陷的改变形式，以及通过寻找这些改变NRII的等位基因特异性抑制因子来研究。为了更精确地确定GlnK在氮调控中的作用，建议进行生理和遗传学实验。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物相互作用的机制。

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.