Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047

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.

M M Garner, A Revzin
PMCID: PMC327330  PMID: 6269071

Abstract

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.

Full text

PDF
3047

Images in this article

3052Image
on p.3052
3053Image
on p.3053
3055Image
on p.3055

Selected References

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

  1. Anthony D. D., Goldthwait D. A. Studies with the RNA polymerase. 3. Enzymatic activity of the monomer form. Biochim Biophys Acta. 1970 Mar 19;204(1):156–167. doi: 10.1016/0005-2787(70)90498-3. [DOI] [PubMed] [Google Scholar]
  2. Boone T., Wilcox G. A rapid high-yield purification procedure for the cyclic adenosine 3',5'-monophosphate receptor protein from Escherichia coli. Biochim Biophys Acta. 1978 Jul 17;541(4):528–534. doi: 10.1016/0304-4165(78)90162-9. [DOI] [PubMed] [Google Scholar]
  3. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  4. Cech C. L., McClure W. R. Characterization of ribonucleic acid polymerase-T7 promoter binary complexes. Biochemistry. 1980 May 27;19(11):2440–2447. doi: 10.1021/bi00552a023. [DOI] [PubMed] [Google Scholar]
  5. Chamberlin M. J., Nierman W. C., Wiggs J., Neff N. A quantitative assay for bacterial RNA polymerases. J Biol Chem. 1979 Oct 25;254(20):10061–10069. [PubMed] [Google Scholar]
  6. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Felsenfeld G., Hirschman S. Z. A neighbor-interaction analysis of the hypochromism and spectra of DNA. J Mol Biol. 1965 Sep;13(2):407–427. doi: 10.1016/s0022-2836(65)80106-1. [DOI] [PubMed] [Google Scholar]
  8. Hardies S. C., Wells R. D. Preparation and characterization of large amounts of restriction fragments containing the E. coli lac control elements. Gene. 1979 Sep;7(1):1–14. doi: 10.1016/0378-1119(79)90039-8. [DOI] [PubMed] [Google Scholar]
  9. Hawley D. K., McClure W. R. In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6381–6385. doi: 10.1073/pnas.77.11.6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection. J Mol Biol. 1972 Sep 28;70(2):157–185. doi: 10.1016/0022-2836(72)90531-1. [DOI] [PubMed] [Google Scholar]
  11. Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. II. The kinetics of the binding reaction. J Mol Biol. 1972 Sep 28;70(2):187–195. doi: 10.1016/0022-2836(72)90532-3. [DOI] [PubMed] [Google Scholar]
  12. Ippen K., Miller J. H., Scaife J., Beckwith J. New controlling element in the Lac operon of E. coli. Nature. 1968 Mar 2;217(5131):825–827. doi: 10.1038/217825a0. [DOI] [PubMed] [Google Scholar]
  13. Jones O. W., Berg P. Studies on the binding of RNA polymerase to polynucleotides. J Mol Biol. 1966 Dec 28;22(2):199–209. doi: 10.1016/0022-2836(66)90126-4. [DOI] [PubMed] [Google Scholar]
  14. Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
  15. Maizels N. M. The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3585–3589. doi: 10.1073/pnas.70.12.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Majors J. Initiation of in vitro mRNA synthesis from the wild-type lac promoter. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4394–4398. doi: 10.1073/pnas.72.11.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Majors J. Specific binding of CAP factor to lac promoter DNA. Nature. 1975 Aug 21;256(5519):672–674. doi: 10.1038/256672a0. [DOI] [PubMed] [Google Scholar]
  18. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  19. McClure W. R. Rate-limiting steps in RNA chain initiation. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5634–5638. doi: 10.1073/pnas.77.10.5634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pastan I., Adhya S. Cyclic adenosine 5'-monophosphate in Escherichia coli. Bacteriol Rev. 1976 Sep;40(3):527–551. doi: 10.1128/br.40.3.527-551.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Revzin A., Woychik R. P. Quantitation of the interaction of EScherichia coli RNA polymerase holoenzyme with double-helical DNA using a thermodynamically rigorous centrifugation method. Biochemistry. 1981 Jan 20;20(2):250–256. doi: 10.1021/bi00505a004. [DOI] [PubMed] [Google Scholar]
  22. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  23. Riggs A. D., Bourgeois S., Newby R. F., Cohn M. DNA binding of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):365–368. doi: 10.1016/0022-2836(68)90261-1. [DOI] [PubMed] [Google Scholar]
  24. Riggs A. D., Reiness G., Zubay G. Purification and DNA-binding properties of the catabolite gene activator protein. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1222–1225. doi: 10.1073/pnas.68.6.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Saxe S. A., Revzin A. Cooperative binding to DNA of catabolite activator protein of Escherichia coli. Biochemistry. 1979 Jan 23;18(2):255–263. doi: 10.1021/bi00569a003. [DOI] [PubMed] [Google Scholar]
  27. Seeburg P. H., Nüsslein C., Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd. Eur J Biochem. 1977 Mar 15;74(1):107–113. doi: 10.1111/j.1432-1033.1977.tb11372.x. [DOI] [PubMed] [Google Scholar]
  28. Silverstone A. E., Arditti R. R., Magasanik B. Catabolite-insensitive revertants of lac promoter mutants. Proc Natl Acad Sci U S A. 1970 Jul;66(3):773–779. doi: 10.1073/pnas.66.3.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takahashi M., Blazy B., Baudras A. Non-specific interactions of CRP from E. coli with native and denatured DNAs: control of binding by cAMP and cGMP and by cation concentration. Nucleic Acids Res. 1979 Nov 24;7(6):1699–1712. doi: 10.1093/nar/7.6.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Walter G., Zillig W., Palm P., Fuchs E. Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem. 1967 Dec;3(2):194–201. doi: 10.1111/j.1432-1033.1967.tb19515.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES