INTERACTIONS OF THE SIGNAL TRANSDUCTION PROTEIN GLNK

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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.