Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli.
Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRII (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRII, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase glnAp2 promoter.
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
J
J
Feng
M R
MR
Atkinson
W
W
McCleary
J B
JB
Stock
B L
BL
Wanner
A J
AJ
Ninfa
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D001426:Bacterial Proteins; D002221:Carbamyl Phosphate / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis*; D008954:Models, Biological; D009154:Mutation; D010755:Organophosphates / Q000494:pharmacology; D051922:PII Nitrogen Regulatory Proteins; D010756:Phosphoric Acids / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000577:Amides; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010755:Organophosphates; D051922:PII Nitrogen Regulatory Proteins; D010756:Phosphoric Acids; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C011632:acetyl phosphate; D002221:Carbamyl Phosphate; D000255:Adenosine Triphosphate; C011067:phosphoramidic acid; D000251:Adenosine Triphosphatases; D005974:Glutamate-Ammonia Ligase