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. 1992 Oct 11;20(19):5035–5039. doi: 10.1093/nar/20.19.5035

Specificities of three tight-binding Lac repressors.

P Kolkhof 1
PMCID: PMC334280  PMID: 1408819

Abstract

Tight binding mutants of Lac repressor exhibit complex repression phenomena. In this work, in vivo Lac operator binding of three such mutants of E. coli Lac repressor (X86: ser 61-leu, l12: pro 3-tyr and the double mutant l12X86: pro 3-tyr, ser 61-leu) was analyzed. Repression of beta-galactosidase synthesis controlled by ideal lac operator and its 27 symmetric operator variants containing each possible base-pair at each single half-operator position in the presence of the tight-binding Lac repressor mutants was determined. The average increase of repression with all operator variants was about 3 fold with the X86 mutant. It was about 4 fold with the l12 mutant and about 2 fold with the double mutant l12X86 as compared to wildtype Lac repressor. The X86 mutant showed the same increase of affinity to all operator variants, whereas the l12 and l12X86 mutants exhibited lower repression with some variants than with most others. These results suggest that the X86 mutant has gained no additional specificity. In contrast the l12 mutant and the l12X86 mutant exhibit a relaxed specificity for certain base pairs in positions 1 and 3 of lac operator. This suggests that the extreme N-terminus of Lac repressor may interact with the inner base-pairs in the minor groove.

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Selected References

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

  1. Adler K., Beyreuther K., Fanning E., Geisler N., Gronenborn B., Klemm A., Müller-Hill B., Pfahl M., Schmitz A. How lac repressor binds to DNA. Nature. 1972 Jun 9;237(5354):322–327. doi: 10.1038/237322a0. [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. Betz J. L. Affinities of tight-binding lactose repressors for wild-type and pseudo-operators. J Mol Biol. 1987 Jun 5;195(3):495–504. doi: 10.1016/0022-2836(87)90178-1. [DOI] [PubMed] [Google Scholar]
  4. Betz J. L., Sadler J. R. Tight-binding repressors of the lactose operon. J Mol Biol. 1976 Aug 5;105(2):293–319. doi: 10.1016/0022-2836(76)90113-3. [DOI] [PubMed] [Google Scholar]
  5. Boelens R., Scheek R. M., van Boom J. H., Kaptein R. Complex of lac repressor headpiece with a 14 base-pair lac operator fragment studied by two-dimensional nuclear magnetic resonance. J Mol Biol. 1987 Jan 5;193(1):213–216. doi: 10.1016/0022-2836(87)90638-3. [DOI] [PubMed] [Google Scholar]
  6. Carey J. trp repressor arms contribute binding energy without occupying unique locations on DNA. J Biol Chem. 1989 Feb 5;264(4):1941–1945. [PubMed] [Google Scholar]
  7. Chamness G. C., Willson C. D. An unusual lac repressor mutant. J Mol Biol. 1970 Nov 14;53(3):561–565. doi: 10.1016/0022-2836(70)90084-7. [DOI] [PubMed] [Google Scholar]
  8. Clarke N. D., Beamer L. J., Goldberg H. R., Berkower C., Pabo C. O. The DNA binding arm of lambda repressor: critical contacts from a flexible region. Science. 1991 Oct 11;254(5029):267–270. doi: 10.1126/science.254.5029.267. [DOI] [PubMed] [Google Scholar]
  9. Ebright R. H. Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator. Proc Natl Acad Sci U S A. 1986 Jan;83(2):303–307. doi: 10.1073/pnas.83.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eliason J. L., Weiss M. A., Ptashne M. NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2339–2343. doi: 10.1073/pnas.82.8.2339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  12. Hurlburt B. K., Yanofsky C. The NH2-terminal arms of trp repressor participate in repressor/operator association. Nucleic Acids Res. 1992 Jan 25;20(2):337–341. doi: 10.1093/nar/20.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jobe A., Bourgeois S. The lac repressor-operator interaction. VII. A repressor with unique binding properties: the X86 repressor. J Mol Biol. 1972 Dec 14;72(1):139–152. doi: 10.1016/0022-2836(72)90075-7. [DOI] [PubMed] [Google Scholar]
  14. Jordan S. R., Pabo C. O. Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions. Science. 1988 Nov 11;242(4880):893–899. doi: 10.1126/science.3187530. [DOI] [PubMed] [Google Scholar]
  15. Kleina L. G., Miller J. H. Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors. J Mol Biol. 1990 Mar 20;212(2):295–318. doi: 10.1016/0022-2836(90)90126-7. [DOI] [PubMed] [Google Scholar]
  16. Kolkhof P., Teichmann D., Kisters-Woike B., von Wilcken-Bergmann B., Müller-Hill B. Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator. EMBO J. 1992 Aug;11(8):3031–3038. doi: 10.1002/j.1460-2075.1992.tb05373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lamerichs R. M., Boelens R., Van der Marel G. A., Van Boom J. H., Kaptein R. Assignment of the 1H-NMR spectrum of a lac repressor headpiece-operator complex in H2O and identification of NOEs. Consequences for protein-DNA interaction. Eur J Biochem. 1990 Dec 12;194(2):629–637. doi: 10.1111/j.1432-1033.1990.tb15662.x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. MONOD J., CHANGEUX J. P., JACOB F. Allosteric proteins and cellular control systems. J Mol Biol. 1963 Apr;6:306–329. doi: 10.1016/s0022-2836(63)80091-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Nelson H. C., Sauer R. T. Interaction of mutant lambda repressors with operator and non-operator DNA. J Mol Biol. 1986 Nov 5;192(1):27–38. doi: 10.1016/0022-2836(86)90461-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  25. Pfahl M. lac Repressor-operator interaction. Analysis of the X86 repressor mutant. J Mol Biol. 1976 Sep 25;106(3):857–869. doi: 10.1016/0022-2836(76)90269-2. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schmitz A., Coulondre C., Miller J. H. Genetic studies of the lac repressor. V. Repressors which bind operator more tightly generated by suppression and reversion of nonsense mutations. J Mol Biol. 1978 Aug 15;123(3):431–454. doi: 10.1016/0022-2836(78)90089-x. [DOI] [PubMed] [Google Scholar]

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