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
. 1985 Apr;82(8):2339–2343. doi: 10.1073/pnas.82.8.2339

NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations.

J L Eliason, M A Weiss, M Ptashne
PMCID: PMC397553  PMID: 3157988

Abstract

Several lines of evidence indicate that the phage lambda repressor recognizes its operator by using, in part, an alpha helix (the "recognition helix"), which it inserts into the major groove of DNA. In addition to its recognition helix, lambda repressor has an "arm," consisting of the first six amino acids, that wraps around the DNA helix. We constructed plasmids that, in Escherichia coli, direct the expression of derivatives of lambda repressor that lack the NH2-terminal one, three, six, or seven amino acids. We studied these modified proteins in vivo and in vitro, and from our results we argue that the arm: contributes a large portion of the binding energy; helps to determine sequence specificity of binding and, in particular, the relative affinities for two wild-type binding sites; determines entirely repressor's response to one operator mutation (a "back-side" mutation); magnifies repressor's response to other operator mutations ("front-side" mutations); and increases the sensitivity of repressor binding to salt concentration and temperature.

Full text

PDF
2340

Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Anderson W. F., Ohlendorf D. H., Takeda Y., Matthews B. W. Structure of the cro repressor from bacteriophage lambda and its interaction with DNA. Nature. 1981 Apr 30;290(5809):754–758. doi: 10.1038/290754a0. [DOI] [PubMed] [Google Scholar]
  3. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blattner F. R., Fiandt M., Hass K. K., Twose P. A., Szybalski W. Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance. Virology. 1974 Dec;62(2):458–471. doi: 10.1016/0042-6822(74)90407-3. [DOI] [PubMed] [Google Scholar]
  5. Brent R., Ptashne M. Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4204–4208. doi: 10.1073/pnas.78.7.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Flashman S. M. Mutational analysis of the operators of bacteriophage lambda. Mol Gen Genet. 1978 Oct 25;166(1):61–73. doi: 10.1007/BF00379730. [DOI] [PubMed] [Google Scholar]
  7. Hecht M. H., Nelson H. C., Sauer R. T. Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding. Proc Natl Acad Sci U S A. 1983 May;80(9):2676–2680. doi: 10.1073/pnas.80.9.2676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Humayun Z., Kleid D., Ptashne M. Sites of contact between lambda operators and lambda repressor. Nucleic Acids Res. 1977;4(5):1595–1607. doi: 10.1093/nar/4.5.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. JACOB F., WOLLMAN E. L. Etude génétique d'un bactériophage tempéré d'Escherichia coli. l. Le système genétique du bactériophage. Ann Inst Pasteur (Paris) 1954 Dec;87(6):653–673. [PubMed] [Google Scholar]
  10. Johnson A. D., Meyer B. J., Ptashne M. Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5061–5065. doi: 10.1073/pnas.76.10.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnson A. D., Pabo C. O., Sauer R. T. Bacteriophage lambda repressor and cro protein: interactions with operator DNA. Methods Enzymol. 1980;65(1):839–856. doi: 10.1016/s0076-6879(80)65078-2. [DOI] [PubMed] [Google Scholar]
  12. Johnson A. D., Poteete A. R., Lauer G., Sauer R. T., Ackers G. K., Ptashne M. lambda Repressor and cro--components of an efficient molecular switch. Nature. 1981 Nov 19;294(5838):217–223. doi: 10.1038/294217a0. [DOI] [PubMed] [Google Scholar]
  13. Johnson A., Meyer B. J., Ptashne M. Mechanism of action of the cro protein of bacteriophage lambda. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1783–1787. doi: 10.1073/pnas.75.4.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lewis M., Jeffrey A., Wang J., Ladner R., Ptashne M., Pabo C. O. Structure of the operator-binding domain of bacteriophage lambda repressor: implications for DNA recognition and gene regulation. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):435–440. doi: 10.1101/sqb.1983.047.01.051. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
  18. Meyer B. J., Maurer R., Ptashne M. Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. J Mol Biol. 1980 May 15;139(2):163–194. doi: 10.1016/0022-2836(80)90303-4. [DOI] [PubMed] [Google Scholar]
  19. Ohlendorf D. H., Anderson W. F., Fisher R. G., Takeda Y., Matthews B. W. The molecular basis of DNA-protein recognition inferred from the structure of cro repressor. Nature. 1982 Aug 19;298(5876):718–723. doi: 10.1038/298718a0. [DOI] [PubMed] [Google Scholar]
  20. Ohlendorf D. H., Anderson W. F., Matthews B. W. Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor. J Mol Evol. 1983;19(2):109–114. doi: 10.1007/BF02300748. [DOI] [PubMed] [Google Scholar]
  21. Pabo C. O., Krovatin W., Jeffrey A., Sauer R. T. The N-terminal arms of lambda repressor wrap around the operator DNA. Nature. 1982 Jul 29;298(5873):441–443. doi: 10.1038/298441a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Pabo C. O., Sauer R. T., Sturtevant J. M., Ptashne M. The lambda repressor contains two domains. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1608–1612. doi: 10.1073/pnas.76.4.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Record M. T., Jr, deHaseth P. L., Lohman T. M. Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry. 1977 Nov 1;16(22):4791–4796. doi: 10.1021/bi00641a005. [DOI] [PubMed] [Google Scholar]
  25. Roberts T. M., Kacich R., Ptashne M. A general method for maximizing the expression of a cloned gene. Proc Natl Acad Sci U S A. 1979 Feb;76(2):760–764. doi: 10.1073/pnas.76.2.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sauer R. T., Pabo C. O., Meyer B. J., Ptashne M., Backman K. C. Regulatory functions of the lambda repressor reside in the amino-terminal domain. Nature. 1979 May 31;279(5712):396–400. doi: 10.1038/279396a0. [DOI] [PubMed] [Google Scholar]
  27. Sauer R. T., Yocum R. R., Doolittle R. F., Lewis M., Pabo C. O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature. 1982 Jul 29;298(5873):447–451. doi: 10.1038/298447a0. [DOI] [PubMed] [Google Scholar]
  28. Schwarz E., Scherer G., Hobom G., Kössel H. Nucleotide sequence of cro, cII and part of the O gene in phage lambda DNA. Nature. 1978 Mar 30;272(5652):410–414. doi: 10.1038/272410a0. [DOI] [PubMed] [Google Scholar]
  29. Steitz T. A., Weber I. T., Matthew J. B. Catabolite gene activator protein: structure, homology with other proteins, and cyclic AMP and DNA binding. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):419–426. doi: 10.1101/sqb.1983.047.01.049. [DOI] [PubMed] [Google Scholar]
  30. Weiss M. A., Eliason J. L., States D. J. Dynamic filtering by two-dimensional 1H NMR with application to phage lambda repressor. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6019–6023. doi: 10.1073/pnas.81.19.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weiss M. A., Karplus M., Patel D. J., Sauer R. T. Solution NMR studies of intact lambda repressor. J Biomol Struct Dyn. 1983 Oct;1(1):151–157. doi: 10.1080/07391102.1983.10507431. [DOI] [PubMed] [Google Scholar]
  32. Weiss M. A., Sauer R. T., Patel D. J., Karplus M. Amino-terminal arm of the lambda repressor: a 1H NMR study. Biochemistry. 1984 Oct 23;23(22):5090–5095. doi: 10.1021/bi00317a002. [DOI] [PubMed] [Google Scholar]
  33. Wharton R. P., Brown E. L., Ptashne M. Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor. Cell. 1984 Sep;38(2):361–369. doi: 10.1016/0092-8674(84)90491-4. [DOI] [PubMed] [Google Scholar]
  34. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [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