The NRI/NRII two-component regulatory system of Escherichia coli regulates the expression of genes involved in nitrogen assimilation in response to signals of carbon and nitrogen status. These signals regulate the activity of the PH and GInK signal transduction proteins, which is turn regulate the kinase and phosphatase activites of NIRII. The kinase and phosphatase activies of NRII control the phosphorylation state of NRI, which is only able to activate transcription when phosphorylated. We propose to study the mechanism and regulation of the kinase and phosphatase activities of NRH. These studies should advance our understanding of metabolic regulation in E. coli and the mechanisms of signal transduction by the two-component regulatory systems. Four main approaches are proposed. First, the structure of NRJI and domains derivied from NRII will be investigated, along with the complex of P11 and NRII and the complex of PH with the isolated kinase domain of NRII. Second, the mechanisms responsible for the effect of mutations altering the kinase and phosphatase activities of NIR11 will be investigated. These studies will involve biochemical approaches using the isolated central domain of NRH and full-length mutant versions of NRII, as well as genetic studies of the regulation of the phosphatase activity. Third, studies with intact cells will be used to characterize the binding of PIT and G1nK to the kinase domain of NRII. Fourth, the epistasis relationships of mutations affecting the kinase and phosphatase activities of NRII will be investigated, and the ability of mutations affecting the phosphatase activity to complement one another will be investigated. Together, these approaches should result in a mechanistic understanding of the NRII kinase and phosphatase activities and their regulation by PH and G1nK.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059637-03
Application #
6636320
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Shapiro, Bert I
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
3
Fiscal Year
2003
Total Cost
$281,299
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Jiang, Peng; Ninfa, Alexander J (2011) A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli. Biochemistry 50:10929-40
Jiang, Peng; Ventura, Alejandra C; Sontag, Eduardo D et al. (2011) Load-induced modulation of signal transduction networks. Sci Signal 4:ra67
Ninfa, Alexander J (2010) Use of two-component signal transduction systems in the construction of synthetic genetic networks. Curr Opin Microbiol 13:240-5
Ventura, Alejandra C; Jiang, Peng; Van Wassenhove, Lauren et al. (2010) Signaling properties of a covalent modification cycle are altered by a downstream target. Proc Natl Acad Sci U S A 107:10032-7
Jiang, Peng; Ninfa, Alexander J (2009) Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer. Biochemistry 48:11522-31
Jiang, Peng; Ninfa, Alexander J (2009) Alpha-ketoglutarate controls the ability of the Escherichia coli PII signal transduction protein to regulate the activities of NRII (NrB but does not control the binding of PII to NRII. Biochemistry 48:11514-21
Jiang, Peng; Ninfa, Alexander J (2007) Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 46:12979-96
Jiang, Peng; Mayo, Avraham E; Ninfa, Alexander J (2007) Escherichia coli glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49): kinetic characterization of regulation by PII, PII-UMP, glutamine, and alpha-ketoglutarate. Biochemistry 46:4133-46
Jiang, Peng; Pioszak, Augen A; Ninfa, Alexander J (2007) Structure-function analysis of glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49) of Escherichia coli. Biochemistry 46:4117-32
Ninfa, Alexander J (2007) Regulation of carbon and nitrogen metabolism: adding regulation of ion channels and another second messenger to the mix. Proc Natl Acad Sci U S A 104:4243-4

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