The Escherichia coli glnB gene encodes a central component of nitrogen regulation known as PII. PII interacts with three different proteins. The product of glnD, a bifunctional uridylyltransferase (UT) and uridylyl-removing enzyme (UR) catalyzes the uridylylation of PII when nitrogen is scarce and catalyzes the deuridylylation of PII-UMP when nitrogen is plentiful. Both the uridylylated and unmodified forms of PII also interact with the adenylyltransferase enzyme (ATase) that covalently modifies glutamine synthetase in response to nitrogen availability; PII stimulates the adenylylation (inactivation) of glutamine synthetase by the ATase, and PII-UMP stimulates the de-adenylylation (activation) of glutamine synthetase by the ATase. Finally, the unmodified form of PII interacts with the bifunctional kinase/phosphatase protein NRII, product of glnL (ntrB), eliciting a phosphatase activity that results in the dephosphorylation of the transcriptional activator NRI-Phosphate by the NRII/Pll phosphatase. This event in turn causes transcription at sigma 54-dependant nitrogen regulated promoters to cease. We propose to study the protein-protein interactions of the PII protein and its receptor proteins using genetic and biochemical methods. We describe a plan for the isolation and rapid sequencing of many glnB mutations that specifically destroy either the interaction with NRII or the interaction with the UT/UR. In addition, we describe genetic and biochemical methods designed to probe the PII-NRII interaction. The results of our study should help to elucidate the nature of the interactions between PII and the three other proteins that it contacts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047460-05
Application #
2184894
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1992-05-01
Project End
1996-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109