GlnGPhosphorylation

GlnGPhosphorylation - Proc.Nati.Acad Sci.USA...

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Unformatted text preview: Proc.Nati.Acad. Sci.USA Vol.83,pp.5909-5913,August1986 Biochemistry CovalentmodificationoftheginGproduct, NRI, bytheglnL product, NRII, regulatesthetranscriptionoftheglnALG operoninEscherichiacoli (glutaminesynthetase/phosphorylation/nitrogenmetabolism/positivecontrol) ALEXANDER J.NINFA AND BORISMAGASANIK* DepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,MA 02139 ContributedbyBorisMagasanik,May5,1986 ABSTRACT Transcriptionfrom nitrogen-regulated pro- moters,suchasglnAp2,requirestheginG gene product, NRI, aswellastherpoN(glnF)geneproduct,aor,andis regulated by theglnLgeneproduct, NR1I. We findthatina reaction mixturecontaining NRI,NRII,andATP, NRII catalyzesthe transfer ofthe y phosphate ofATP to NRI. Thiscovalent modificationof NRI occursconcurrentlywiththeacquisitionof the abilityby the reactionmixture toactivatetranscription fromglnAp2.Inthepresenceof PI,, theproductofglnB, NRI catalyzestheremovalofthephosphatefrom NRI-phosphate. Thisreactionoccursconcurrentlywiththelossbythereaction mixtureoftheabilitytoactivatetranscriptionfromglnAp2.On the basisofthisevidence, we propose that NRI-phosphate activatestranscriptionfromnitrogen-regulatedpromotersand thattheroleofNRIIiscontroloftheformationandbreakdown of NRI-phosphate inresponsetocellularsignalsofnitrogen availability. InEscherichiacoliandotherentericbacteria,transcriptionof theglnALG operon,whichcontainsthestructuralgenefor glutamine synthetase (glnA), is activated in response to nitrogenstarvationatthepromoter ginAp2(1).Thisactiva- tionrequiresthe DNA-bindingproteinNRI,theproductof ginG, as well as o-, the product ofrpoN(glnF), and is regulatedby NRII,theproductofginL (2-8).Wild-typecells are able to decrease or increase very rapidly the rate of transcriptioninitiationatglnAp2inresponsetotheaddition or removal ofammonia. Mutants thatlack NRII lackthe abilityforthisrapidresponse. Nonetheless, thesemutants haveregulatedlevelsofnitrogen-regulatedgeneproductsin thesteadystate,indicatingthata slowerandlessefficient NRII-independentmechanismforthe regulationoftranscrip- tionfromglnAp2doesexist. The regulationofginAexpressionby NRIIrequiresthe productsoftwoadditionalgenes, ginDandginB (9,10).The ginDgeneproductisa uridylyltransferase(UTase)required for the conversion of PI,, the ginB gene product, to a uridylylatedform, and auridylyl-removingenzyme, which catalyzes the reverse reaction. The ability of UTase to convert PI, to PII-UMPisstimulatedby2-ketoglutarateand, conversely, theabilityofuridylyl-removingenzyme tore- move the uridylyl group from PII-UMP isstimulated by glutamine(11).Thus,ammoniastarvation,whichresultsina highintracellularratioof2-ketoglutaratetoglutamine,causes the conversion of PI, to PII-UMP. Growth with ammonia excess,whichresultsinahighintracellularratioofglutamine to2-ketoglutarate,causestheconversionof PII-UMPto PI,....
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GlnGPhosphorylation - Proc.Nati.Acad Sci.USA...

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