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Transcription from nitrogen-regulated pro- NRII-independent mechanism for the regulation of transcription from glnAp2 does exist.The regulation of ginA expression by NRII requires the products of two additional genes, ginD and ginB (9, 10). The ginD gene product is a uridylyltransferase (UTase) required for the conversion of PI,, the ginB gene product, to a uridylylated form, and a uridylyl-removing enzyme, which catalyzes the reverse reaction. The ability of UTase to convert PI, to PII-UMP is stimulated by 2-ketoglutarate and, conversely, the ability of uridylyl-removing enzyme to remove the uridylyl group from PII-UMP is stimulated by glutamine (11). Thus, ammonia starvation, which results in a high intracellular ratio of 2-ketoglutarate to glutamine, causes the conversion of PI, to PII-UMP. Growth

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It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of ginA, the structural gene for glutamine synthetase, and that NRxI catalyzes the transfer of the -phosphate ofATP to NRI. We r-Phosphohistidine (histidine 3-phosphate) was synthesized by the reaction of histidine with the potassium salt of phosphoramidate (4,5). The latter was synthesized according to Sheridan et al. (6).Purified Proteins. The source of NRI was a 20-liter culture of E. coli strain TH19/pTH806 grown at 370C as previously described (1). Sonication, streptomycin sulfate precipitation, and ammonium sulfate precipitation were done as previously described (7) For sodium borohydride reduction, NRI was phosphorylated in a final volume of 1.25 ml and loaded on a 1.5-ml heparin-Sepharose column equilibrated in 50 mM Tris-HCl, pH 8, washed with 7 ml of 50 mM Tris.HCl/1 mM EDTA, pH 8, and eluted with 10 ml of 50 mM Tris.HCl/1 mM EDTA/1 M NaCl. The fractions containing phosphorylated NRI (3 ml) were pooled. Phosphorylated NRI was precipitated on ice with 12 ml of 5% trichloroacetic acid, washed with 12 ml of ethanol/diethyl ether (1:1, vol/vol), and evaporated to dryness.Isolation ofNRII-Phosphate. NRI, (7 (14) and Nishigaki et al. (9) was used with minor modifications as described in the legend to Fig. 7.Identification of Phosphohistidine in NRH-Phosphate. This was done as described by Smith et al. (10) with minor modifications as described in the legend to Fig. 4. Phosphohistidine was detected after two-dimensional chromatography by spraying the plates with 0.1 M HCI and heating at 100'C for 15 min, followed by treatment with the Pauly diazo reagent. NaDodSO4/PAGE was done as described by Laemmli (11). RESULTS Phosphorylation of NRu. The incubation of NRII with ATP resulted in the transfer of the y-phosphate of ATP to NRII. In the experiments illustrated in Fig. 1, reaction mixtures containing NRII and ATP labeled with 32P in the y position were spotted on nitrocellulose paper filters, which were then washed with Tris*HCl buffer to remove the nucleotides.Incorporation of radioactivity occurred only when ATP was labeled in the y position (Fig. 1), not when it was labeled in the a position (data not shown). The transfer ofthe phosphate to NRII, which required a divalent cation such as Mg2+, proceeded rapidly at 370C and more slowly at 0C. The