Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Mar;87(5):1744–1748. doi: 10.1073/pnas.87.5.1744

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

T Heyduk 1, J C Lee 1
PMCID: PMC53559  PMID: 2155424

Abstract

A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.

Full text

PDF
1744

Selected References

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

  1. Baudras A., Blazy B., Takahashi M. Association spécifique du complexe CRP-AMP-cyclique sur la région de contrôle de l'opéron lactose d'Escherichia coli K 12: une étude fluorimétrique directe utilisant des fragments de DNA de différentes longueurs. Biochimie. 1983 Jul;65(7):437–440. doi: 10.1016/s0300-9084(83)80063-7. [DOI] [PubMed] [Google Scholar]
  2. Brown A. M., Crothers D. M. Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7387–7391. doi: 10.1073/pnas.86.19.7387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chu B. C., Orgel L. E. Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds. Nucleic Acids Res. 1988 May 11;16(9):3671–3691. doi: 10.1093/nar/16.9.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Draper D. E., von Hippel P. H. Measurement of macromolecular equilibrium binding constants by a sucrose gradient band sedimentation method. Application to protein-nucleic acid interactions. Biochemistry. 1979 Mar 6;18(5):753–760. doi: 10.1021/bi00572a003. [DOI] [PubMed] [Google Scholar]
  5. Frankel A. D., Ackers G. K., Smith H. O. Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease. Biochemistry. 1985 Jun 4;24(12):3049–3054. doi: 10.1021/bi00333a037. [DOI] [PubMed] [Google Scholar]
  6. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garner M. M., Revzin A. Stoichiometry of catabolite activator protein/adenosine cyclic 3',5'-monophosphate interactions at the lac promoter of Escherichia coli. Biochemistry. 1982 Nov 23;21(24):6032–6036. doi: 10.1021/bi00267a001. [DOI] [PubMed] [Google Scholar]
  9. Heyduk T., Lee J. C. Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities. Biochemistry. 1989 Aug 22;28(17):6914–6924. doi: 10.1021/bi00443a021. [DOI] [PubMed] [Google Scholar]
  10. Jensen D. E., von Hippel P. H. A boundary sedimentation velocity method for determining nonspecific nucleic acid-protein interaction binding parameters. Anal Biochem. 1977 May 15;80(1):267–281. doi: 10.1016/0003-2697(77)90645-5. [DOI] [PubMed] [Google Scholar]
  11. Kolb A., Spassky A., Chapon C., Blazy B., Buc H. On the different binding affinities of CRP at the lac, gal and malT promoter regions. Nucleic Acids Res. 1983 Nov 25;11(22):7833–7852. doi: 10.1093/nar/11.22.7833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McSwiggen J. A., Bear D. G., von Hippel P. H. Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies. J Mol Biol. 1988 Feb 20;199(4):609–622. doi: 10.1016/0022-2836(88)90305-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  15. Schmitz A. Cyclic AMP receptor proteins interacts with lactose operator DNA. Nucleic Acids Res. 1981 Jan 24;9(2):277–292. doi: 10.1093/nar/9.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Senear D. F., Brenowitz M., Shea M. A., Ackers G. K. Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding. Biochemistry. 1986 Nov 18;25(23):7344–7354. doi: 10.1021/bi00371a016. [DOI] [PubMed] [Google Scholar]
  17. Sippel T. O. Microfluorometric analysis of protein thiol groups with a coumarinylphenylmaleimide. J Histochem Cytochem. 1981 Dec;29(12):1377–1381. doi: 10.1177/29.12.7320496. [DOI] [PubMed] [Google Scholar]
  18. Straney D. C., Straney S. B., Crothers D. M. Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J Mol Biol. 1989 Mar 5;206(1):41–57. doi: 10.1016/0022-2836(89)90522-6. [DOI] [PubMed] [Google Scholar]
  19. Takahashi M., Blazy B., Baudras A. An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli. Biochemistry. 1980 Oct 28;19(22):5124–5130. doi: 10.1021/bi00563a029. [DOI] [PubMed] [Google Scholar]
  20. Takahashi M., Blazy B., Baudras A., Hillen W. Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets. J Mol Biol. 1989 Jun 20;207(4):783–796. doi: 10.1016/0022-2836(89)90244-1. [DOI] [PubMed] [Google Scholar]
  21. Teare J., Wollenzien P. Specificity of site directed psoralen addition to RNA. Nucleic Acids Res. 1989 May 11;17(9):3359–3372. doi: 10.1093/nar/17.9.3359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES