Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1987 May;6(5):1481–1491. doi: 10.1002/j.1460-2075.1987.tb02390.x

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

H Krämer, M Niemöller, M Amouyal, B Revet, B von Wilcken-Bergmann, B Müller-Hill
PMCID: PMC553955  PMID: 3301328

Abstract

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.

Full text

PDF
1483

Images in this article

Selected References

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

  1. Abermann R., Bahl C. P., Marians K. J., Salpeter M. M., Wu R. Studies on the lactose operon. III. Visualization and physical mapping of the lactose repressor-operator complex. J Mol Biol. 1976 Jan 5;100(1):109–114. doi: 10.1016/s0022-2836(76)80038-1. [DOI] [PubMed] [Google Scholar]
  2. Besse M., von Wilcken-Bergmann B., Müller-Hill B. Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter. EMBO J. 1986 Jun;5(6):1377–1381. doi: 10.1002/j.1460-2075.1986.tb04370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  4. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  5. Culard F., Maurizot J. C. Lac repressor - lac operator interaction. Circular dichroism study. Nucleic Acids Res. 1981 Oct 10;9(19):5175–5184. doi: 10.1093/nar/9.19.5175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dandanell G., Hammer K. Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3333–3338. doi: 10.1002/j.1460-2075.1985.tb04085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drew H. R., Travers A. A. DNA bending and its relation to nucleosome positioning. J Mol Biol. 1985 Dec 20;186(4):773–790. doi: 10.1016/0022-2836(85)90396-1. [DOI] [PubMed] [Google Scholar]
  8. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  9. Dunn T. M., Hahn S., Ogden S., Schleif R. F. An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5017–5020. doi: 10.1073/pnas.81.16.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fried M. G., Crothers D. M. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J Mol Biol. 1984 Jan 25;172(3):263–282. doi: 10.1016/s0022-2836(84)80026-1. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fritz H. J., Bicknäse H., Gleumes B., Heibach C., Rosahl S., Ehring R. Characterization of two mutations in the Escherichia coli galE gene inactivating the second galactose operator and comparative studies of repressor binding. EMBO J. 1983;2(12):2129–2135. doi: 10.1002/j.1460-2075.1983.tb01713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Griffith J., Hochschild A., Ptashne M. DNA loops induced by cooperative binding of lambda repressor. Nature. 1986 Aug 21;322(6081):750–752. doi: 10.1038/322750a0. [DOI] [PubMed] [Google Scholar]
  16. Herrin G. L., Jr, Bennett G. N. Role of DNA regions flanking the tryptophan promoter of Escherichia coli. II. Insertion of lac operator fragments. Gene. 1984 Dec;32(3):349–356. doi: 10.1016/0378-1119(84)90010-6. [DOI] [PubMed] [Google Scholar]
  17. Hochschild A., Ptashne M. Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix. Cell. 1986 Mar 14;44(5):681–687. doi: 10.1016/0092-8674(86)90833-0. [DOI] [PubMed] [Google Scholar]
  18. Horowitz D. S., Wang J. C. Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling. J Mol Biol. 1984 Feb 15;173(1):75–91. doi: 10.1016/0022-2836(84)90404-2. [DOI] [PubMed] [Google Scholar]
  19. Irani M. H., Orosz L., Adhya S. A control element within a structural gene: the gal operon of Escherichia coli. Cell. 1983 Mar;32(3):783–788. doi: 10.1016/0092-8674(83)90064-8. [DOI] [PubMed] [Google Scholar]
  20. Kania J., Brown D. T. The functional repressor parts of a tetrameric lac repressor-beta-galactosidase chimaera are organized as dimers. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3529–3533. doi: 10.1073/pnas.73.10.3529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kania J., Müller-Hill B. Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules. Eur J Biochem. 1977 Oct 3;79(2):381–386. doi: 10.1111/j.1432-1033.1977.tb11819.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Koo H. S., Wu H. M., Crothers D. M. DNA bending at adenine . thymine tracts. Nature. 1986 Apr 10;320(6062):501–506. doi: 10.1038/320501a0. [DOI] [PubMed] [Google Scholar]
  24. Kuhnke G., Krause A., Heibach C., Gieske U., Fritz H. J., Ehring R. The upstream operator of the Escherichia coli galactose operon is sufficient for repression of transcription initiated at the cyclic AMP-stimulated promoter. EMBO J. 1986 Jan;5(1):167–173. doi: 10.1002/j.1460-2075.1986.tb04192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Majumdar A., Adhya S. Demonstration of two operator elements in gal: in vitro repressor binding studies. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6100–6104. doi: 10.1073/pnas.81.19.6100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martin K., Huo L., Schleif R. F. The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3654–3658. doi: 10.1073/pnas.83.11.3654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McKay D. B., Pickover C. A., Steitz T. A. Escherichia coli lac repressor is elongated with its operator DNA binding domains located at both ends. J Mol Biol. 1982 Mar 25;156(1):175–183. doi: 10.1016/0022-2836(82)90465-x. [DOI] [PubMed] [Google Scholar]
  29. Mossing M. C., Record M. T., Jr Thermodynamic origins of specificity in the lac repressor-operator interaction. Adaptability in the recognition of mutant operator sites. J Mol Biol. 1985 Nov 20;186(2):295–305. doi: 10.1016/0022-2836(85)90106-8. [DOI] [PubMed] [Google Scholar]
  30. Mossing M. C., Record M. T., Jr Upstream operators enhance repression of the lac promoter. Science. 1986 Aug 22;233(4766):889–892. doi: 10.1126/science.3090685. [DOI] [PubMed] [Google Scholar]
  31. Müller-Hill B. Lac repressor and lac operator. Prog Biophys Mol Biol. 1975;30(2-3):227–252. doi: 10.1016/0079-6107(76)90011-0. [DOI] [PubMed] [Google Scholar]
  32. O'Gorman R. B., Dunaway M., Matthews K. S. DNA binding characteristics of lactose repressor and the trypsin-resistant core repressor. J Biol Chem. 1980 Nov 10;255(21):10100–10106. [PubMed] [Google Scholar]
  33. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  34. Reznikoff W. S., Winter R. B., Hurley C. K. The location of the repressor binding sites in the lac operon. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2314–2318. doi: 10.1073/pnas.71.6.2314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Sadler J. R., Sasmor H., Betz J. L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6785–6789. doi: 10.1073/pnas.80.22.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shore D., Baldwin R. L. Energetics of DNA twisting. I. Relation between twist and cyclization probability. J Mol Biol. 1983 Nov 15;170(4):957–981. doi: 10.1016/s0022-2836(83)80198-3. [DOI] [PubMed] [Google Scholar]
  38. Shore D., Baldwin R. L. Energetics of DNA twisting. II. Topoisomer analysis. J Mol Biol. 1983 Nov 15;170(4):983–1007. doi: 10.1016/s0022-2836(83)80199-5. [DOI] [PubMed] [Google Scholar]
  39. Shore D., Langowski J., Baldwin R. L. DNA flexibility studied by covalent closure of short fragments into circles. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4833–4837. doi: 10.1073/pnas.78.8.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Simons A., Tils D., von Wilcken-Bergmann B., Müller-Hill B. Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1624–1628. doi: 10.1073/pnas.81.6.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stueber D., Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. doi: 10.1002/j.1460-2075.1982.tb01329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Takahashi K., Vigneron M., Matthes H., Wildeman A., Zenke M., Chambon P. Requirement of stereospecific alignments for initiation from the simian virus 40 early promoter. Nature. 1986 Jan 9;319(6049):121–126. doi: 10.1038/319121a0. [DOI] [PubMed] [Google Scholar]
  43. Tuggle C. K., Fuchs J. A. Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control. EMBO J. 1986 May;5(5):1077–1085. doi: 10.1002/j.1460-2075.1986.tb04325.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ulanovsky L., Bodner M., Trifonov E. N., Choder M. Curved DNA: design, synthesis, and circularization. Proc Natl Acad Sci U S A. 1986 Feb;83(4):862–866. doi: 10.1073/pnas.83.4.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ullmann A. One-step purification of hybrid proteins which have beta-galactosidase activity. Gene. 1984 Jul-Aug;29(1-2):27–31. doi: 10.1016/0378-1119(84)90162-8. [DOI] [PubMed] [Google Scholar]
  46. Valentin-Hansen P., Albrechtsen B., Løve Larsen J. E. DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J. 1986 Aug;5(8):2015–2021. doi: 10.1002/j.1460-2075.1986.tb04458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Whitson P. A., Olson J. S., Matthews K. S. Thermodynamic analysis of the lactose repressor-operator DNA interaction. Biochemistry. 1986 Jul 1;25(13):3852–3858. doi: 10.1021/bi00361a017. [DOI] [PubMed] [Google Scholar]
  48. Winter R. B., von Hippel P. H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 2. The Escherichia coli repressor--operator interaction: equilibrium measurements. Biochemistry. 1981 Nov 24;20(24):6948–6960. doi: 10.1021/bi00527a029. [DOI] [PubMed] [Google Scholar]
  49. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES