Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Mar 15;26(6):1373–1381. doi: 10.1093/nar/26.6.1373

DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy.

F W Sevenich 1, J Langowski 1, V Weiss 1, K Rippe 1
PMCID: PMC147426  PMID: 9490780

Abstract

Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA.

Full Text

The Full Text of this article is available as a PDF (158.4 KB).

Selected References

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

  1. Ackers G. K., Shea M. A., Smith F. R. Free energy coupling within macromolecules. The chemical work of ligand binding at the individual sites in co-operative systems. J Mol Biol. 1983 Oct 15;170(1):223–242. doi: 10.1016/s0022-2836(83)80234-4. [DOI] [PubMed] [Google Scholar]
  2. Blattner F. R., Plunkett G., 3rd, Bloch C. A., Perna N. T., Burland V., Riley M., Collado-Vides J., Glasner J. D., Rode C. K., Mayhew G. F. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5;277(5331):1453–1462. doi: 10.1126/science.277.5331.1453. [DOI] [PubMed] [Google Scholar]
  3. Chen P., Reitzer L. J. Active contribution of two domains to cooperative DNA binding of the enhancer-binding protein nitrogen regulator I (NtrC) of Escherichia coli: stimulation by phosphorylation and the binding of ATP. J Bacteriol. 1995 May;177(9):2490–2496. doi: 10.1128/jb.177.9.2490-2496.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eigen M., Rigler R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5740–5747. doi: 10.1073/pnas.91.13.5740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Farez-Vidal M. E., Wilson T. J., Davidson B. E., Howlett G. J., Austin S., Dixon R. A. Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC. Mol Microbiol. 1996 Dec;22(5):779–788. doi: 10.1046/j.1365-2958.1996.01530.x. [DOI] [PubMed] [Google Scholar]
  7. Flashner Y., Weiss D. S., Keener J., Kustu S. Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain. J Mol Biol. 1995 Jun 16;249(4):700–713. doi: 10.1006/jmbi.1995.0330. [DOI] [PubMed] [Google Scholar]
  8. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  9. Heyduk T., Lee J. C. Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1744–1748. doi: 10.1073/pnas.87.5.1744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heyduk T., Lee J. C., Ebright Y. W., Blatter E. E., Zhou Y., Ebright R. H. CAP interacts with RNA polymerase in solution in the absence of promoter DNA. Nature. 1993 Aug 5;364(6437):548–549. doi: 10.1038/364548a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Kinjo M., Rigler R. Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy. Nucleic Acids Res. 1995 May 25;23(10):1795–1799. doi: 10.1093/nar/23.10.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klingler J., Friedrich T. Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy. Biophys J. 1997 Oct;73(4):2195–2200. doi: 10.1016/S0006-3495(97)78251-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klose K. E., Weiss D. S., Kustu S. Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. J Mol Biol. 1993 Jul 5;232(1):67–78. doi: 10.1006/jmbi.1993.1370. [DOI] [PubMed] [Google Scholar]
  15. LeTilly V., Royer C. A. Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding. Biochemistry. 1993 Aug 3;32(30):7753–7758. doi: 10.1021/bi00081a021. [DOI] [PubMed] [Google Scholar]
  16. Lohman T. M., Mascotti D. P. Thermodynamics of ligand-nucleic acid interactions. Methods Enzymol. 1992;212:400–424. doi: 10.1016/0076-6879(92)12026-m. [DOI] [PubMed] [Google Scholar]
  17. Maiti S., Haupts U., Webb W. W. Fluorescence correlation spectroscopy: diagnostics for sparse molecules. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11753–11757. doi: 10.1073/pnas.94.22.11753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mettke I., Fiedler U., Weiss V. Mechanism of activation of a response regulator: interaction of NtrC-P dimers induces ATPase activity. J Bacteriol. 1995 Sep;177(17):5056–5061. doi: 10.1128/jb.177.17.5056-5061.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moore J. B., Shiau S. P., Reitzer L. J. Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli. J Bacteriol. 1993 May;175(9):2692–2701. doi: 10.1128/jb.175.9.2692-2701.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. North A. K., Klose K. E., Stedman K. M., Kustu S. Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: the puzzle of nitrogen regulatory protein C. J Bacteriol. 1993 Jul;175(14):4267–4273. doi: 10.1128/jb.175.14.4267-4273.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pan C. Q., Finkel S. E., Cramton S. E., Feng J. A., Sigman D. S., Johnson R. C. Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. J Mol Biol. 1996 Dec 13;264(4):675–695. doi: 10.1006/jmbi.1996.0669. [DOI] [PubMed] [Google Scholar]
  23. Porter S. C., North A. K., Wedel A. B., Kustu S. Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. Genes Dev. 1993 Nov;7(11):2258–2273. doi: 10.1101/gad.7.11.2258. [DOI] [PubMed] [Google Scholar]
  24. Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
  25. Record M. T., Jr, Lohman M. L., De Haseth P. Ion effects on ligand-nucleic acid interactions. J Mol Biol. 1976 Oct 25;107(2):145–158. doi: 10.1016/s0022-2836(76)80023-x. [DOI] [PubMed] [Google Scholar]
  26. Reedstrom R. J., Brown M. P., Grillo A., Roen D., Royer C. A. Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides. J Mol Biol. 1997 Oct 31;273(3):572–585. doi: 10.1006/jmbi.1997.1333. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Rippe K., Guthold M., von Hippel P. H., Bustamante C. Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. J Mol Biol. 1997 Jul 11;270(2):125–138. doi: 10.1006/jmbi.1997.1079. [DOI] [PubMed] [Google Scholar]
  30. Rippe K., Ramsing N. B., Jovin T. M. Spectroscopic properties and helical stabilities of 25-nt parallel-stranded linear DNA duplexes. Biochemistry. 1989 Nov 28;28(24):9536–9541. doi: 10.1021/bi00450a043. [DOI] [PubMed] [Google Scholar]
  31. Royer C. A., Beechem J. M. Numerical analysis of binding data: advantages, practical aspects, and implications. Methods Enzymol. 1992;210:481–505. doi: 10.1016/0076-6879(92)10025-9. [DOI] [PubMed] [Google Scholar]
  32. Royer C. A. Improvements in the numerical analysis of thermodynamic data from biomolecular complexes. Anal Biochem. 1993 Apr;210(1):91–97. doi: 10.1006/abio.1993.1155. [DOI] [PubMed] [Google Scholar]
  33. Révet B., Brahms S., Brahms G. Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7535–7539. doi: 10.1073/pnas.92.16.7535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sterrer S., Henco K. Fluorescence correlation spectroscopy (FCS)--a highly sensitive method to analyze drug/target interactions. J Recept Signal Transduct Res. 1997 Jan-May;17(1-3):511–520. doi: 10.3109/10799899709036624. [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. Vámosi G., Gohlke C., Clegg R. M. Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes. Biophys J. 1996 Aug;71(2):972–994. doi: 10.1016/S0006-3495(96)79300-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Weglenski P., Ninfa A. J., Ueno-Nishio S., Magasanik B. Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I. J Bacteriol. 1989 Aug;171(8):4479–4485. doi: 10.1128/jb.171.8.4479-4485.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wyman C., Rombel I., North A. K., Bustamante C., Kustu S. Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science. 1997 Mar 14;275(5306):1658–1661. doi: 10.1126/science.275.5306.1658. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES