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• W2103095934 abstract "Nitrogen limitation in Escherichia coli activates about 100 genes. Their expression requires the response regulator NtrC (also called nitrogen regulator I or NRI). Phosphorylation of the amino-terminal domain (NTD) of NtrC activates the neighboring central domain and leads to transcriptional activation from promoters that require σ54-containing RNA polymerase. The NTD has five β strands alternating with five α helices. Phosphorylation of aspartate 54 has been shown to reposition α helix 3 to β strand 5 (the “3445 face”) within the NTD. To further study the interactions between the amino-terminal and central domains, we isolated strains with alterations in the NTD that were able to grow on a poor nitrogen source in the absence of phosphorylation by the cognate sensor kinase. We identified strains with alterations located in the 3445 face and α helix 5. Both types of alterations stimulated central domain activities. The α helix 5 alterations differed from those in the 3445 face. They did not cause a large scale conformational change in the NTD, which is not necessary for transcriptional activation in these mutants. Yeast two-hybrid analysis indicated that substitutions in both α helix 5 and the 3445 face diminish the interaction between the NTD and the central domain. Our results suggest that α helix 5 of the NTD, in addition to the 3445 face, interacts with the central domain. We present a model of interdomain signal transduction that proposes different functions for α helix 5 and the 3445 face. Nitrogen limitation in Escherichia coli activates about 100 genes. Their expression requires the response regulator NtrC (also called nitrogen regulator I or NRI). Phosphorylation of the amino-terminal domain (NTD) of NtrC activates the neighboring central domain and leads to transcriptional activation from promoters that require σ54-containing RNA polymerase. The NTD has five β strands alternating with five α helices. Phosphorylation of aspartate 54 has been shown to reposition α helix 3 to β strand 5 (the “3445 face”) within the NTD. To further study the interactions between the amino-terminal and central domains, we isolated strains with alterations in the NTD that were able to grow on a poor nitrogen source in the absence of phosphorylation by the cognate sensor kinase. We identified strains with alterations located in the 3445 face and α helix 5. Both types of alterations stimulated central domain activities. The α helix 5 alterations differed from those in the 3445 face. They did not cause a large scale conformational change in the NTD, which is not necessary for transcriptional activation in these mutants. Yeast two-hybrid analysis indicated that substitutions in both α helix 5 and the 3445 face diminish the interaction between the NTD and the central domain. Our results suggest that α helix 5 of the NTD, in addition to the 3445 face, interacts with the central domain. We present a model of interdomain signal transduction that proposes different functions for α helix 5 and the 3445 face. Escherichia coli can utilize a variety of compounds as sole nitrogen sources. Ammonia supports rapid growth, whereas alternate nitrogen sources, such as amino acids, result in slower nitrogen-limited growth. The response to nitrogen limitation, known as the nitrogen-regulated (Ntr) 1The abbreviations used are: Ntr, nitrogen-regulated; NTD, aminoterminal domain; HSQC, heteronuclear single quantum correlation; NtrC-DE, NtrC-D109N/E110K.1The abbreviations used are: Ntr, nitrogen-regulated; NTD, aminoterminal domain; HSQC, heteronuclear single quantum correlation; NtrC-DE, NtrC-D109N/E110K. response, affects the synthesis of about 100 proteins that assimilate ammonia, transport several amino acids and other nitrogenous compounds, and degrade a few nitrogen-containing compounds (1.Zimmer D.P. Soupene E. Lee H.L. Wendisch V.F. Khodursky A.B. Peter B.J. Bender R.A. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14674-14679Crossref PubMed Scopus (283) Google Scholar, 2.Reitzer L. Schneider B.L. Microbiol. Mol. Biol. Rev. 2001; 65: 422-444Crossref PubMed Scopus (220) Google Scholar). Intracellular glutamine is the primary effector of the Ntr response (3.Ikeda T.P. Shauger A.E. Kustu S. J. Mol. Biol. 1996; 259: 589-607Crossref PubMed Scopus (154) Google Scholar). With high glutamine (i.e. in nitrogen-rich media) NtrC (also called nitrogen regulator I or NRI) is unmodified, which prevents expression of Ntr genes. In contrast, low glutamine (i.e. in nitrogen-limited media) leads to phosphorylation of NtrC and activation of Ntr genes (4.Ninfa A.J. Atkinson M.R. Kamberov E.S. Feng J. Ninfa E.G. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 67-88Google Scholar, 5.Magasanik B. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 1344-1356Google Scholar, 6.Reitzer L.J. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 391-407Google Scholar). For example, NtrC-P activates the glnALG operon, which specifies glutamine synthetase, NtrB (also called nitrogen regulator II or NRII), and NtrC, respectively (6.Reitzer L.J. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 391-407Google Scholar). NtrC activates Ntr genes from enhancer sites, which often consist of two adjacent NtrC binding sites located up to 250 base pairs away from the transcription start site (7.Reitzer L.J. Magasanik B. Cell. 1986; 45: 785-792Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 8.Kiupakis A.K. Reitzer L. J. Bacteriol. 2002; 184: 2940-2950Crossref PubMed Scopus (65) Google Scholar). Unphosphorylated NtrC in solution is a dimer, but DNA-bound NtrC-P forms either a hexamer or octamer (9.Wyman C. Rombel I. North A.K. Bustamante C. Kustu S. Science. 1997; 275: 1658-1661Crossref PubMed Scopus (209) Google Scholar, 10.Rippe K. Mucke N. Schulz A. J. Mol. Biol. 1998; 278: 915-933Crossref PubMed Scopus (54) Google Scholar, 11.Rombel I. North A. Hwang I. Wyman C. Kustu S. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 157-166Crossref PubMed Scopus (81) Google Scholar). The enhancer increases the local concentration of NtrC and serves as a template for oligomerization (12.Wedel A. Weiss D.S. Popham D. Droge P. Kustu S. Science. 1990; 248: 486-490Crossref PubMed Scopus (151) Google Scholar, 13.Porter S.C. North A.K. Wedel A.B. Kustu S. Genes Dev. 1993; 7: 2258-2273Crossref PubMed Scopus (156) Google Scholar). From the enhancer, NtrC interacts with σ54-containing RNA polymerase and hydrolyzes ATP to provide the energy needed to catalyze open complex formation (5.Magasanik B. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 1344-1356Google Scholar, 7.Reitzer L.J. Magasanik B. Cell. 1986; 45: 785-792Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 14.Ninfa A.J. Reitzer L.J. Magasanik B. Cell. 1987; 50: 1039-1046Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 15.Popham D.L. Szeto D. Keener J. Kustu S. Science. 1989; 243: 629-635Crossref PubMed Scopus (315) Google Scholar, 16.Reitzer L.J. Movsas B. Magasanik B. J. Bacteriol. 1989; 171: 5512-5522Crossref PubMed Google Scholar, 17.Porter S.C. North A.K. Kustu S. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 147-158Google Scholar, 18.Wedel A. Kustu S. Genes Dev. 1995; 9: 2042-2052Crossref PubMed Scopus (139) Google Scholar). NtrC is a multidomain response regulator that, like similar proteins, contains conserved receiver and output domains. NtrC also has a carboxyl-terminal domain required for dimerization and binding to DNA. The 124-residue amino-terminal domain (NTD) contains the site of phosphorylation, aspartate 54, and binds Mg2+, which is essential for phosphorylation and dephosphorylation (19.Sanders D.A. Gillece-Castro B.L. Burlingame A.L. Koshland Jr, D.E. J. Bacteriol. 1992; 174: 5117-5122Crossref PubMed Google Scholar, 20.Stock A.M. Martinez-Hackert E. Rasmussen B.F. West A.H. Stock J.B. Ringe D. Petsko G.A. Biochemistry. 1993; 32: 13375-13380Crossref PubMed Scopus (197) Google Scholar). The phosphorylated form of NtrC has a half-life of around 4 min (21.Keener J. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4976-4980Crossref PubMed Scopus (233) Google Scholar, 22.Weiss V. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8919-8923Crossref PubMed Scopus (186) Google Scholar). In the absence of its cognate sensor kinase, small phosphate-containing molecules (e.g. acetyl phosphate) can phosphorylate NtrC (23.Lukat G.S. McCleary W.R. Stock A.M. Stock J.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 718-722Crossref PubMed Scopus (412) Google Scholar). Some of these small phosphodonors may have physiologically relevant functions (23.Lukat G.S. McCleary W.R. Stock A.M. Stock J.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 718-722Crossref PubMed Scopus (412) Google Scholar, 24.Schneider B.L. Shiau S.P. Reitzer L.J. J. Bacteriol. 1991; 173: 6355-6363Crossref PubMed Google Scholar). Structures of the unphosphorylated and phosphorylated receiver domains of NtrC have been determined (25.Volkman B.F. Nohaile M.J. Amy N.K. Kustu S. Wemmer D.E. Biochemistry. 1995; 34: 1413-1424Crossref PubMed Scopus (101) Google Scholar, 26.Kern D. Volkman B.F. Luginbuhl P. Nohaile M.J. Kustu S. Wemmer D.E. Nature. 1999; 402: 894-898Crossref PubMed Scopus (173) Google Scholar, 27.Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14789-14794Crossref PubMed Scopus (122) Google Scholar, 28.Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2450) Google Scholar) (Fig. 1). Five parallel β strands alternate with five α helices (25.Volkman B.F. Nohaile M.J. Amy N.K. Kustu S. Wemmer D.E. Biochemistry. 1995; 34: 1413-1424Crossref PubMed Scopus (101) Google Scholar). The solvent-exposed loops that join the units of secondary structure contain not only the site of phosphorylation (between β3 and α3) but also other conserved residues that are spatially located near the site of phosphorylation and comprise the phosphorylation active site: the β1-α1, β4-α4, and β5-α5 loops. Upon phosphorylation, the region bounded by α helix 3 and β strand 5 is displaced with respect to the remainder of the NTD. This region has been termed the 3445 face (for α3, β/α4, and β5). A major characteristic of this conformational change is the axial rotation of α helix 4 by about 100° leading to a shift of two amino acids in the helix register and causing the hydrophobic surface of the helix, which is buried when the NTD is unphosphorylated, to become solvent-exposed. Furthermore the loop containing the site of phosphorylation and the loops flanking α4 appear to take on a stable conformation not seen in the inactive form of the domain (26.Kern D. Volkman B.F. Luginbuhl P. Nohaile M.J. Kustu S. Wemmer D.E. Nature. 1999; 402: 894-898Crossref PubMed Scopus (173) Google Scholar). This phosphorylation-induced structural change is responsible for the altered interaction with the central domain that leads to activation of transcription (29.Hwang I. Thorgeirsson T. Lee J. Kustu S. Shin Y.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4880-4885Crossref PubMed Scopus (37) Google Scholar, 30.Lee J. Owens J.T. Hwang I. Meares C. Kustu S. J. Bacteriol. 2000; 182: 5188-5195Crossref PubMed Scopus (38) Google Scholar). All σ54-dependent activators have a domain homologous to the central domain of NtrC (31.Morett E. Segovia L. J. Bacteriol. 1993; 175: 6067-6074Crossref PubMed Google Scholar). This domain interacts with σ54 and couples the energy derived from ATP hydrolysis to formation of open promoter complexes. The central domain of NtrC also contains a component of oligomerization that is required for transcriptional activation (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar, 33.Flashner Y. Weiss D.S. Keener J. Kustu S. J. Mol. Biol. 1995; 249: 700-713Crossref PubMed Scopus (63) Google Scholar). Oligomerization leads to increased stability of binding to adjacent sites on DNA, which is referred to as cooperative binding. Phosphorylation of the NTD stimulates this oligomerization (13.Porter S.C. North A.K. Wedel A.B. Kustu S. Genes Dev. 1993; 7: 2258-2273Crossref PubMed Scopus (156) Google Scholar, 32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar, 33.Flashner Y. Weiss D.S. Keener J. Kustu S. J. Mol. Biol. 1995; 249: 700-713Crossref PubMed Scopus (63) Google Scholar, 34.Weiss V. Claverie-Martin F. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5088-5092Crossref PubMed Scopus (115) Google Scholar), which, in turn, stimulates ATP hydrolysis. Phosphorylation does not affect activities of the carboxyl-terminal domain: dimerization and binding to a single site on DNA (13.Porter S.C. North A.K. Wedel A.B. Kustu S. Genes Dev. 1993; 7: 2258-2273Crossref PubMed Scopus (156) Google Scholar, 34.Weiss V. Claverie-Martin F. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5088-5092Crossref PubMed Scopus (115) Google Scholar, 35.Klose K.E. North A.K. Stedman K.M. Kustu S. J. Mol. Biol. 1994; 241: 233-245Crossref PubMed Scopus (57) Google Scholar). Our aim was to analyze the interaction between the NTD and the central domain of NtrC. We isolated and characterized mutants with alterations in the NTD that could activate nitrogen-regulated genes without NtrB, i.e. variants in which the NTD is apparently sending a phosphorylation-independent activation signal to the central domain. Altered residues were found not only in the 3445 face but also in α helix 5. We show that the alterations in α helix 5, unlike those in the 3445 face, do not affect the overall structure of the NTD. However, two-hybrid interaction analysis indicated that alterations in helix 5 impair domain-domain interactions. We propose that α helix 5, in addition to the 3445 face, interacts with the central domain and that the function of this interaction may be to inhibit phosphorylation-dependent oligomerization. Bacterial Strains—All strains used for mutant isolation and assay of glutamine synthetase were derivatives of W3110 (lacIq lacL8). The additional genotypes are as follows: LR1 (ntrC10::Tn5), LR20 (ntrC10::Tn5/λgln107), SPS1 (ntrC10::Tn5 glnD99::Tn10), and SN24 (ΔntrBC) (24.Schneider B.L. Shiau S.P. Reitzer L.J. J. Bacteriol. 1991; 173: 6355-6363Crossref PubMed Google Scholar, 36.Shiau S.P. Chen P. Reitzer L.J. J. Bacteriol. 1993; 175: 190-199Crossref PubMed Google Scholar). Cell Growth—The minimal growth medium contained W salts (37.Rothstein D.M. Pahel G. Tyler B. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7372-7376Crossref PubMed Scopus (39) Google Scholar), 0.4% carbon source, 0.2% nitrogen source, and 0.02% thiamine. Enzyme Assays—Assays for glutamine synthetase and β-galactosidase were performed as described previously (37.Rothstein D.M. Pahel G. Tyler B. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7372-7376Crossref PubMed Scopus (39) Google Scholar, 38.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-355Google Scholar). Activity units are nmol of product min–1 mg of protein–1. The ATPase assay was based on measurement of ADP (39.Lowry O.H. Passonneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, New York1972: 147-149Google Scholar). A 100-μl reaction mixture contained 50 mm Tris-HCl (pH 7.5), 100 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol, 0.1 mm EDTA, 1.3 mm phosphoenol-pyruvate, 35 units of lactate dehydrogenase (rabbit muscle), 50 units of pyruvate kinase (rabbit muscle), 0.2 mm NADH, and 500 nm NtrC. 200 nm NtrB was present to phosphorylate NtrC. When DNA was added, 10 nm supercoiled pVW7, which contains NtrC binding sites 1 and 2 upstream of the glnA promoter, was used (34.Weiss V. Claverie-Martin F. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5088-5092Crossref PubMed Scopus (115) Google Scholar). The reaction mixtures were incubated for 10 min at 37 °C, and ATP (final concentration, 1 mm) was added to start the reaction. The change of NADH concentration was measured spectrophotometrically at 340 nm. Units of ATPase activity are mol of ATP min–1 mol of NtrC monomer–1. Mutagenesis and Mutant Isolation—The template used for mutagenesis of the DNA coding for the NTD of NtrC was pXY12, which is essentially pBLS1 (24.Schneider B.L. Shiau S.P. Reitzer L.J. J. Bacteriol. 1991; 173: 6355-6363Crossref PubMed Google Scholar) except for an ApaI site. This site was created by changing DNA for codons 135 and 136 from GGCCCA to GGGCCC, which introduces two silent mutations. PCR-based mutagenesis of the NTD of NtrC was accomplished as described previously except that 0.1 μm MnCl2 and 4 mm MgCl2 were used (40.Leung D.M. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar). The PCR product was digested by restriction enzymes PflMI and ApaI (from codons 17 to 136) and subcloned back into pXY12. The mutagenized plasmid was transformed into strain SN24 and plated on glucose-arginine minimal medium. The colonies were inoculated in liquid overnight cultures, and the plasmids were purified and retransformed into SN24. The transformants were retested for their phenotype. The ntrC alleles were sequenced using the Sequenase kit from U.S. Biochemical Corp. Proteins—Full-length variants were overexpressed and purified as described previously (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar, 41.Moore J.B. Shiau S.P. Reitzer L.J. J. Bacteriol. 1993; 175: 2692-2701Crossref PubMed Google Scholar). An unusual property of NtrC-D109N/E110K was its precipitation between 20 and 30% (NH4)2SO4 saturation. It was 90% pure at this stage. Wild-type NtrC, NtrB, and σ54 were purified as described previously (41.Moore J.B. Shiau S.P. Reitzer L.J. J. Bacteriol. 1993; 175: 2692-2701Crossref PubMed Google Scholar). DNase I Footprinting—The method was basically as described previously (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar). A 32P-labeled EcoRI-PstI fragment (153 bp) from pVW7 was used for binding of NtrC to sites 1 and 2. A 75-μl reaction mixture contained 35 mm Tris acetate (pH 7.9), 70 mm K+-acetate, 5 mm Mg2+-acetate, 19 mmNH4+-acetate, 0.1 mg/ml bovine serum albumin, 0.7 mm dithiothreitol, 3 μg of sonicated salmon sperm DNA, 5% glycerol, and 0.5 nm DNA. NtrC concentrations were as shown in the figures. NtrC and DNA were incubated for 10 min at 37 °C, and then a predetermined dilution of DNase I was added to the reaction. The reaction was incubated for another 7 min and stopped by adding 37.5 μl of a stop buffer containing 8 mNH4+-acetate and 250 μg/ml yeast tRNA. The mixture was extracted with phenol-chloroform and precipitated with ethanol. The samples were run on an 8% polyacrylamide gel containing 8 m urea. The protected bands were quantified using a PhosphorImager (Amersham Biosciences). The quantitation of occupancy has been described previously (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar). In Vitro Phosphorylation and Dephosphorylation—42.5 μl of solution A (40 nm NtrB, 1× transcription buffer (see below), 1 mg/ml bovine serum albumin, and 15 μCi of [γ-32P]ATP) and 40 μl of solution B (4 μm of NtrC, 1× transcription buffer) were separately preincubated at 37 °C. A 2.5-μl aliquot of solution A was mixed with an equal volume of a pH 6.8 stop buffer (10 mm EDTA, 2% SDS, 0.6% Tris-HCl, 10% glycerol, 0.1% dithiothreitol, 0.01% bromphenol blue) and put on ice. The remaining 40 μl of solution A was then mixed with solution B. 10-μl samples were taken at 0.5 and 2.5 min after mixing. At 3 min after mixing, 20 μl of unlabeled ATP in 1× transcription buffer was added to the reaction (giving a final unlabeled ATP concentration of 2.5 mm). Samples were taken at various times and loaded on a 10% polyacrylamide gel containing SDS without heating. The gel was placed in a PhosphorImager cassette, and the bands were quantified using a PhosphorImager program (Amersham Biosciences). In Vitro Transcription—The basic procedures have been described previously (42.Ninfa A.J. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5909-5913Crossref PubMed Scopus (386) Google Scholar). In the first step, a 25-μl mixture containing 1× transcription buffer (50 mm Tris acetate, pH 8, 100 mm K+-acetate, 8 mm Mg2+-acetate, 27 mmNH4+-acetate, 3.5% polyethylene glycol, 1 mm dithiothreitol); either supercoiled plasmid pTH8, which carries the glnA promoter (43.Hunt T.P. Magasanik B. Proc. Natl. Acad. Sc. i U. S. A. 1985; 82: 8453-8457Crossref PubMed Scopus (188) Google Scholar), or pAK10, which carries the ast promoter (8.Kiupakis A.K. Reitzer L. J. Bacteriol. 2002; 184: 2940-2950Crossref PubMed Scopus (65) Google Scholar); 1 unit of RNase inhibitor (Eppendorf-5 Prime, Inc.); E. coli RNA core polymerase (Epicenter Technologies); and σ54 protein were incubated for 5 min at 37 °C. An NtrC variant (1 μl) was then added. When NtrC was phosphorylated, 1 μl of NtrB was added, and the mixture was incubated for 5 min at 37 °C. To permit the formation of open complexes, 2 μlof50mm ATP was added to the transcription buffer, and the mixture was incubated for 10 min. For the final step, a 7-μl mixture containing 500 μg of heparin; a 1.8 mm concentration of GTP, CTP, and UTP; and 10 μCi of [α-32P]UTP in transcription buffer was added into the reaction. The final concentrations of components were 10 nm DNA, 100 nm core RNA polymerase, 300 nm σ54, 100 nm NtrC for reactions with NtrB or 300 nm NtrC for reactions without NtrB, 100 nm NtrB (when added), 4 mm ATP, 500 μm GTP, 500 μm CTP, and 100 μm UTP. After 10 min, the 25-μl reaction was stopped by adding 25 μl of a stop buffer containing 50 mm EDTA and 100 μg/ml yeast tRNA. The sample was extracted with phenol, and 40 μl was mixed with 15 μl of a loading dye mixture containing 95% formamide, 20 mm EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF. The sample was heated at 90 °C for 3 min, and 30 μl was subjected to electrophoresis in a 5% polyacrylamide, 8 m urea gel. Expression and Purification of Histidine-tagged NTD of NtrC for NMR—Plasmid pACH6 is a derivative of pRSET-A (New England Biolabs) that expresses the NTD of NtrC (residues 1–140) with a hexahistidine tag under the control of a T7 promoter. E. coli strain BL21λ was transformed with pACH6 expressing wild-type or variant NTDs, and the proteins were overexpressed essentially as described previously (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar). Cell pellets from overexpression were disrupted by sonication, and the purification protocol was as described for batch binding purification under native conditions (Qiagen “The QIAexpressionist” Handbook) with the following exception. To provide a higher stringency for binding histidine-tagged protein, the lysis and wash buffers contained 20 and 50 mm imidazole, respectively. Proteins were concentrated (Centricon system) and dialyzed into a 50 mm phosphate buffer, pH 6.75 that contained 150 mm NaCl. Expression and Purification of Native NTD of NtrC for NMR—Plasmid pACH12 codes for the NTD of NtrC (residues 1–124) fused to the Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein from pTYB11 under T7 control (Impact CN system, New England Biolabs). Autocatalytic cleavage at the junction site of NtrC and the intein allows for purification of the native 124-residue NTD. Overexpression was performed in E. coli strain ER2566 from New England Biolabs. Overexpression and purification were as described for the New England Biolabs Impact CN system. NMR Spectroscopy—Proteins were purified as described above, and NMR spectroscopy was carried out on a 500-MHz Varian Unity-INOVA spectrometer with z axis gradient in the Department of Chemistry at The University of Texas at Dallas. 1H-15N heteronuclear single quantum correlation (HSQC) experiments used a sensitivity-enhanced pulse sequence (44.Zhang O. Kay L.E. Olivier J.P. Forman-Kay J.D. J. Biomol. NMR. 1994; 4: 845-858Crossref PubMed Scopus (612) Google Scholar) and were performed at 25 °C with sweep widths of 6100 Hz for 1H and 1400 Hz for 15N. All HSQC experiments were collected with 64 complex points in the indirect dimension. The Yeast Two-hybrid System—The plasmids pAS1-CYH2 and pACTII and strain Y190 (MATa leu2–3,112, ura3–52, trp1–901, his3-Δ200, ade2–101, gal4Δ, gal80Δ, URA3::GAL-lacZ, LYS2::GAL-HIS3, cyhr) used for the yeast two-hybrid system are slightly modified versions of pAS1, pACT, and Y153 (45.Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1298) Google Scholar). For the plasmids coding for the NTD of NtrC fused to the GAL4 DNA-binding domain, the plasmid pT7-NcoI-NT was used. pT7-NcoI-NT is the derivative of pCB5, which contains an ntrC gene with a deletion of the central domain coding region under the control of a phage T7 promoter (32.Chen P. Reitzer L.J. J. Bacteriol. 1995; 177: 2490-2496Crossref PubMed Google Scholar). An NheI linker (CTAGCTAGCTAG), which contains a stop codon in all three reading frames, was inserted at an engineered SalI site at codon 142 of the glnG gene in pCB5. A unique NcoI site was also created at the translational start site of the glnG gene. A 0.8-kb NcoI-BamHI fragment of pT7-NcoI-NT, which codes for an NtrC with a deletion of the central domain, with stop codons at the end of the NTD was then ligated into the polylinker site of pAS1-CYH2, producing pAS1-CYH2-NTA. This plasmid contains the gene encoding a fusion of the DNA-binding domain of GAL4 to the NTD of NtrC. After construction, the fusion junctions were checked by sequencing. For the central domain fusion construct, the plasmid pXY15BNN was used. pXY15BNN is a derivative of pXY12 with three linkers inserted at different positions. An NheI linker (with stop codons in three reading frames) was inserted at a ScaI site within glnG gene (codon 387). An NcoI linker was inserted at an ApaI site (codon 136). A BamHI linker was inserted at an SspI site 500 bp downstream of the gene. Then the 1.3-kb NcoI-BamHI fragment of pXY15BNN was cloned into the polylinker site of pACTII to produce the final construct, pACTII-CEN, which contains the gene encoding a fusion of the GAL4 activation domain to the central domain of NtrC. The plasmids were then transformed into yeast strain Y190 as described previously (46.Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1773) Google Scholar). The transformants were plated on synthetic dextrose medium (0.15% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, and 2% dextrose) supplemented with 0.15 mm adenine and 3 mm histidine. The colonies were then tested for growth on the same medium with 30 mm 3-amino-1,2,4-triazole instead of histidine. The assay for β-galactosidase in yeast was performed as described previously (47.Hannig E.M. Hinnebusch A.G. Mol. Cell Biol. 1988; 8: 4808-4820Crossref PubMed Scopus (56) Google Scholar). The plasmids used for the positive controls, pACT4 and pSUG1, were kindly provided by Stephen Johnston (The University of Texas Southwestern Medical Center). ACT4 has previously been found to interact with SUG1. 2S. Johnston, personal communication. Phosphorylation of the NTD of NtrC controls central domain-mediated oligomerization and ATP hydrolysis, which are required for transcriptional activation. To analyze the communication between the amino-terminal and central domains, we sought mutants with alterations in the NTD that could activate Ntr genes without NtrB. We used a previously described procedure: selection for derivatives of an ntrB mutant that could grow with arginine as sole nitrogen source (33.Flashner Y. Weiss D.S. Keener J. Kustu S. J. Mol. Biol. 1995; 249: 700-713Crossref PubMed Scopus (63) Google Scholar). Arginine utilization requires high expression of the glnALG operon, which codes for the ammonia-assimilating glutamine synthetase, NtrB, and NtrC, respectively, and expression of the astCADBE operon, whose products degrade arginine. glnALG expression requires NtrC phosphorylation by either NtrB-P or acetyl phosphate. In contrast, astCADBE expression requires NtrC phosphorylation by both NtrB-P and acetyl phosphate (24.Schneider B.L. Shiau S.P. Reitzer L.J. J. Bacteriol. 1991; 173: 6355-6363Crossref PubMed Google Scholar, 48.Atkinson M.R. Blauwkamp T.A. Bondarenko V. Studitsky V. Ninfa A.J. J. Bacteriol. 2002; 184: 5358-5363Crossref PubMed Scopus (70) Google Scholar). In other words, glnALG is expressed in a glnL mutant, but astCADBE is not. We selected arginine utilizers from a strain with a deletion of ntrB and ntrC on the chromosome and ntrC expressed on a plasmid. After PCR mutagenesis of ntrC, we subcloned the NTD coding region of NtrC to ensure that the only alterations were within the NTD. We isolated 14 arginine-utilizing mutants with seven different substitutions: R56H, M75I, D86N, D109N, E110K, E116K, and E124K. One derivative had the double substitution D109N/E110K (Fig. 1). Arg-56 is located near the phosphorylation pocket at the carboxyl terminus of β3. Met-75 is in the loop between α3 and β4 on the edge of the β sheet that is opposite the phosphorylation site. As" @default.
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• W2103095934 date "2004-01-01" @default.
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• W2103095934 title "Evidence for a Second Interaction between the Regulatory Amino-terminal and Central Output Domains of the Response Regulator NtrC (Nitrogen Regulator I) in Escherichia coli" @default.
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• W2103095934 doi "https://doi.org/10.1074/jbc.m306181200" @default.
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