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. 1994 Jul 15;13(14):3348–3355. doi: 10.1002/j.1460-2075.1994.tb06637.x

Quality and position of the three lac operators of E. coli define efficiency of repression.

S Oehler 1, M Amouyal 1, P Kolkhof 1, B von Wilcken-Bergmann 1, B Müller-Hill 1
PMCID: PMC395232  PMID: 8045263

Abstract

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

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Selected References

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  1. Adhya S. Multipartite genetic control elements: communication by DNA loop. Annu Rev Genet. 1989;23:227–250. doi: 10.1146/annurev.ge.23.120189.001303. [DOI] [PubMed] [Google Scholar]
  2. Alberti S., Oehler S., von Wilcken-Bergmann B., Krämer H., Müller-Hill B. Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat. New Biol. 1991 Jan;3(1):57–62. [PubMed] [Google Scholar]
  3. Alberti S., Oehler S., von Wilcken-Bergmann B., Müller-Hill B. Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle. EMBO J. 1993 Aug;12(8):3227–3236. doi: 10.1002/j.1460-2075.1993.tb05992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Amouyal M., Mortensen L., Buc H., Hammer K. Single and double loop formation when deoR repressor binds to its natural operator sites. Cell. 1989 Aug 11;58(3):545–551. doi: 10.1016/0092-8674(89)90435-2. [DOI] [PubMed] [Google Scholar]
  5. Amouyal M., von Wilcken-Bergmann B. Repression of the E. coli lactose operon by cooperation between two individually unproductive "half-operator" sites. C R Acad Sci III. 1992;315(11):403–407. [PubMed] [Google Scholar]
  6. Borowiec J. A., Zhang L., Sasse-Dwight S., Gralla J. D. DNA supercoiling promotes formation of a bent repression loop in lac DNA. J Mol Biol. 1987 Jul 5;196(1):101–111. doi: 10.1016/0022-2836(87)90513-4. [DOI] [PubMed] [Google Scholar]
  7. Brenowitz M., Jamison E., Majumdar A., Adhya S. Interaction of the Escherichia coli Gal repressor protein with its DNA operators in vitro. Biochemistry. 1990 Apr 3;29(13):3374–3383. doi: 10.1021/bi00465a033. [DOI] [PubMed] [Google Scholar]
  8. Chuprina V. P., Rullmann J. A., Lamerichs R. M., van Boom J. H., Boelens R., Kaptein R. Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. J Mol Biol. 1993 Nov 20;234(2):446–462. doi: 10.1006/jmbi.1993.1598. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Dandanell G., Valentin-Hansen P., Larsen J. E., Hammer K. Long-range cooperativity between gene regulatory sequences in a prokaryote. 1987 Feb 26-Mar 4Nature. 325(6107):823–826. doi: 10.1038/325823a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Eismann E., von Wilcken-Bergmann B., Müller-Hill B. Specific destruction of the second lac operator decreases repression of the lac operon in Escherichia coli fivefold. J Mol Biol. 1987 Jun 20;195(4):949–952. doi: 10.1016/0022-2836(87)90499-2. [DOI] [PubMed] [Google Scholar]
  13. Freemont P. S., Lane A. N., Sanderson M. R. Structural aspects of protein-DNA recognition. Biochem J. 1991 Aug 15;278(Pt 1):1–23. doi: 10.1042/bj2780001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gho D., Miller J. H. Deletions fusing the i and lac regions of the chromosome in E. coli: isolation and mapping. Mol Gen Genet. 1974;131(2):137–146. doi: 10.1007/BF00266149. [DOI] [PubMed] [Google Scholar]
  16. Haber R., Adhya S. Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9683–9687. doi: 10.1073/pnas.85.24.9683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hopkins J. D. A new class of promoter mutations in the lactose operon of Escherichia coli. J Mol Biol. 1974 Aug 25;87(4):715–724. doi: 10.1016/0022-2836(74)90080-1. [DOI] [PubMed] [Google Scholar]
  18. Kadowaki H., Kadowaki T., Wondisford F. E., Taylor S. I. Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. Gene. 1989 Mar 15;76(1):161–166. doi: 10.1016/0378-1119(89)90018-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Krämer H., Amouyal M., Nordheim A., Müller-Hill B. DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor. EMBO J. 1988 Feb;7(2):547–556. doi: 10.1002/j.1460-2075.1988.tb02844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krämer H., Niemöller M., Amouyal M., Revet B., von Wilcken-Bergmann B., Müller-Hill B. lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J. 1987 May;6(5):1481–1491. doi: 10.1002/j.1460-2075.1987.tb02390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lanzer M., Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. doi: 10.1073/pnas.85.23.8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Law S. M., Bellomy G. R., Schlax P. J., Record M. T., Jr In vivo thermodynamic analysis of repression with and without looping in lac constructs. Estimates of free and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid DNA in vivo. J Mol Biol. 1993 Mar 5;230(1):161–173. doi: 10.1006/jmbi.1993.1133. [DOI] [PubMed] [Google Scholar]
  24. Lehming N., Sartorius J., Niemöller M., Genenger G., v Wilcken-Bergmann B., Müller-Hill B. The interaction of the recognition helix of lac repressor with lac operator. EMBO J. 1987 Oct;6(10):3145–3153. doi: 10.1002/j.1460-2075.1987.tb02625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lehming N., Sartorius J., Oehler S., von Wilcken-Bergmann B., Müller-Hill B. Recognition helices of lac and lambda repressor are oriented in opposite directions and recognize similar DNA sequences. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7947–7951. doi: 10.1073/pnas.85.21.7947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lobell R. B., Schleif R. F. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528–532. doi: 10.1126/science.2237403. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Oehler S., Eismann E. R., Krämer H., Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990 Apr;9(4):973–979. doi: 10.1002/j.1460-2075.1990.tb08199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pabo C. O., Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. doi: 10.1038/298443a0. [DOI] [PubMed] [Google Scholar]
  32. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  33. Pfahl M., Gulde V., Bourgeois S. "Second" and "third operator" of the lac operon: an investigation of their role in the regulatory mechanism. J Mol Biol. 1979 Jan 25;127(3):339–344. doi: 10.1016/0022-2836(79)90333-4. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. SADLER J. R., NOVICK A. THE PROPERTIES OF REPRESSOR AND THE KINETICS OF ITS ACTION. J Mol Biol. 1965 Jun;12:305–327. doi: 10.1016/s0022-2836(65)80255-8. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Schleif R. DNA binding by proteins. Science. 1988 Sep 2;241(4870):1182–1187. doi: 10.1126/science.2842864. [DOI] [PubMed] [Google Scholar]
  39. Schleif R. DNA looping. Annu Rev Biochem. 1992;61:199–223. doi: 10.1146/annurev.bi.61.070192.001215. [DOI] [PubMed] [Google Scholar]
  40. Schmitz A., Galas D. J. The interaction of RNA polymerase and lac repressor with the lac control region. Nucleic Acids Res. 1979 Jan;6(1):111–137. doi: 10.1093/nar/6.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sieg K., Kun J., Pohl I., Scherf A., Müller-Hill B. A versatile phage lambda expression vector system for cloning in Escherichia coli. Gene. 1989 Feb 20;75(2):261–270. doi: 10.1016/0378-1119(89)90272-2. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Straney S. B., Crothers D. M. Lac repressor is a transient gene-activating protein. Cell. 1987 Dec 4;51(5):699–707. doi: 10.1016/0092-8674(87)90093-6. [DOI] [PubMed] [Google Scholar]
  44. 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]

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