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. 1993 May;175(9):2692–2701. doi: 10.1128/jb.175.9.2692-2701.1993

Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli.

J B Moore 1, S P Shiau 1, L J Reitzer 1
PMCID: PMC204572  PMID: 8097516

Abstract

Transcription of many nitrogen-regulated (Ntr) genes requires the phosphorylated form of nitrogen regulator I (NRI, or NtrC), which binds to sites that are analogous to eukaryotic enhancers. A highly conserved regulatory domain contains the site of phosphorylation and controls the function of NRI. We analyzed the effects of substitutions in highly conserved residues that are part of the active site of phosphorylation of NRI in Escherichia coli. Fourteen substitutions of aspartate 54, the site of phosphorylation, impaired the response to nitrogen deprivation. Only one of these variants, NRI D-54-->E (NRI-D54E), could significantly stimulate transcription from glnAp2, the major promoter of the glnALG operon. Cells with this variant grew with arginine as a nitrogen source. Experiments with purified components showed that unphosphorylated NRI-D54E stimulated transcription. In contrast, substitutions at aspartate 11 were not as deleterious as those at aspartate 54. Finally, we showed that NRI-K103R, in which arginine replaces the absolutely conserved lysine, is functionally active and efficiently phosphorylated. This substitution appears to stabilize the phosphoaspartate of NRI. The differences between our results and those from study of homologous proteins suggest that there may be significant differences in the way highly conserved residues participate in the transition to the activated state.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Austin S., Dixon R. The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J. 1992 Jun;11(6):2219–2228. doi: 10.1002/j.1460-2075.1992.tb05281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backman K., Chen Y. M., Magasanik B. Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3743–3747. doi: 10.1073/pnas.78.6.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourret R. B., Borkovich K. A., Simon M. I. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem. 1991;60:401–441. doi: 10.1146/annurev.bi.60.070191.002153. [DOI] [PubMed] [Google Scholar]
  4. Brissette R. E., Tsung K. L., Inouye M. Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli. J Bacteriol. 1991 Jan;173(2):601–608. doi: 10.1128/jb.173.2.601-608.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Contreras A., Drummond M. The effect on the function of the transcriptional activator NtrC from Klebsiella pneumoniae of mutations in the DNA-recognition helix. Nucleic Acids Res. 1988 May 11;16(9):4025–4039. doi: 10.1093/nar/16.9.4025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feng J., Atkinson M. R., McCleary W., Stock J. B., Wanner B. L., Ninfa A. J. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6061–6070. doi: 10.1128/jb.174.19.6061-6070.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirschman J., Wong P. K., Sei K., Keener J., Kustu S. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7525–7529. doi: 10.1073/pnas.82.22.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huala E., Ausubel F. M. The central domain of Rhizobium meliloti NifA is sufficient to activate transcription from the R. meliloti nifH promoter. J Bacteriol. 1989 Jun;171(6):3354–3365. doi: 10.1128/jb.171.6.3354-3365.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huala E., Stigter J., Ausubel F. M. The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol. 1992 Feb;174(4):1428–1431. doi: 10.1128/jb.174.4.1428-1431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hunt T. P., Magasanik B. Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8453–8457. doi: 10.1073/pnas.82.24.8453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jin S. G., Prusti R. K., Roitsch T., Ankenbauer R. G., Nester E. W. Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J Bacteriol. 1990 Sep;172(9):4945–4950. doi: 10.1128/jb.172.9.4945-4950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  14. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lukat G. S., Lee B. H., Mottonen J. M., Stock A. M., Stock J. B. Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis. J Biol Chem. 1991 May 5;266(13):8348–8354. [PubMed] [Google Scholar]
  16. Lukat G. S., Stock A. M., Stock J. B. Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis. Biochemistry. 1990 Jun 12;29(23):5436–5442. doi: 10.1021/bi00475a004. [DOI] [PubMed] [Google Scholar]
  17. Mandecki W. Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7177–7181. doi: 10.1073/pnas.83.19.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ninfa A. J., Ninfa E. G., Lupas A. N., Stock A., Magasanik B., Stock J. Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5492–5496. doi: 10.1073/pnas.85.15.5492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ninfa A. J., Reitzer L. J., Magasanik B. Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers. Cell. 1987 Sep 25;50(7):1039–1046. doi: 10.1016/0092-8674(87)90170-x. [DOI] [PubMed] [Google Scholar]
  21. Ninfa A. J., Ueno-Nishio S., Hunt T. P., Robustell B., Magasanik B. Purification of nitrogen regulator II, the product of the glnL (ntrB) gene of Escherichia coli. J Bacteriol. 1986 Nov;168(2):1002–1004. doi: 10.1128/jb.168.2.1002-1004.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reitzer L. J., Magasanik B. Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1979–1983. doi: 10.1073/pnas.82.7.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reitzer L. J., Magasanik B. Isolation of the nitrogen assimilation regulator NR(I), the product of the glnG gene of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5554–5558. doi: 10.1073/pnas.80.18.5554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reitzer L. J., Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell. 1986 Jun 20;45(6):785–792. doi: 10.1016/0092-8674(86)90553-2. [DOI] [PubMed] [Google Scholar]
  25. Reitzer L. J., Movsas B., Magasanik B. Activation of glnA transcription by nitrogen regulator I (NRI)-phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase. J Bacteriol. 1989 Oct;171(10):5512–5522. doi: 10.1128/jb.171.10.5512-5522.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rothstein D. M., Pahel G., Tyler B., Magasanik B. Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7372–7376. doi: 10.1073/pnas.77.12.7372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanders D. A., Gillece-Castro B. L., Burlingame A. L., Koshland D. E., Jr Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. J Bacteriol. 1992 Aug;174(15):5117–5122. doi: 10.1128/jb.174.15.5117-5122.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanders D. A., Gillece-Castro B. L., Stock A. M., Burlingame A. L., Koshland D. E., Jr Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J Biol Chem. 1989 Dec 25;264(36):21770–21778. [PubMed] [Google Scholar]
  29. Schneider B. L., Shiau S. P., Reitzer L. J. Role of multiple environmental stimuli in control of transcription from a nitrogen-regulated promoter in Escherichia coli with weak or no activator-binding sites. J Bacteriol. 1991 Oct;173(20):6355–6363. doi: 10.1128/jb.173.20.6355-6363.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shiau S. P., Chen P., Reitzer L. J. Effects of insertions and deletions in glnG (ntrC) of Escherichia coli on nitrogen regulator I-dependent DNA binding and transcriptional activation. J Bacteriol. 1993 Jan;175(1):190–199. doi: 10.1128/jb.175.1.190-199.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shiau S. P., Schneider B. L., Gu W., Reitzer L. J. Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth. J Bacteriol. 1992 Jan;174(1):179–185. doi: 10.1128/jb.174.1.179-185.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stock A. M., Mottonen J. M., Stock J. B., Schutt C. E. Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature. 1989 Feb 23;337(6209):745–749. doi: 10.1038/337745a0. [DOI] [PubMed] [Google Scholar]
  33. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  35. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  36. Su W., Porter S., Kustu S., Echols H. DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5504–5508. doi: 10.1073/pnas.87.14.5504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Volz K., Matsumura P. Crystal structure of Escherichia coli CheY refined at 1.7-A resolution. J Biol Chem. 1991 Aug 15;266(23):15511–15519. doi: 10.2210/pdb3chy/pdb. [DOI] [PubMed] [Google Scholar]
  39. Wedel A., Weiss D. S., Popham D., Dröge P., Kustu S. A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science. 1990 Apr 27;248(4954):486–490. doi: 10.1126/science.1970441. [DOI] [PubMed] [Google Scholar]
  40. Weiss D. S., Batut J., Klose K. E., Keener J., Kustu S. The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription. Cell. 1991 Oct 4;67(1):155–167. doi: 10.1016/0092-8674(91)90579-n. [DOI] [PubMed] [Google Scholar]
  41. Weiss V., Claverie-Martin F., Magasanik B. Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5088–5092. doi: 10.1073/pnas.89.11.5088. [DOI] [PMC free article] [PubMed] [Google Scholar]

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