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
. 1977 Nov;74(11):4973–4976. doi: 10.1073/pnas.74.11.4973

Contacts between the lac repressor and the thymines in the lac operator.

R Ogata, W Gilbert
PMCID: PMC432080  PMID: 270732

Abstract

We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.

Full text

PDF
4973

Images in this article

4974Image
on p.4974

Selected References

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

  1. 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]
  2. Bahl C. P., Wu R., Stawinsky J., Narang S. A. Minimal length of the lactose operator sequence for the specific recognition by the lactose repressor. Proc Natl Acad Sci U S A. 1977 Mar;74(3):966–970. doi: 10.1073/pnas.74.3.966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Couch J. L., Hanawalt P. C. Analysis of 5-bromouracil distribution in partially substituted deoxyribonucleic acids. Anal Biochem. 1971 May;41(1):51–56. doi: 10.1016/0003-2697(71)90190-4. [DOI] [PubMed] [Google Scholar]
  4. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hassur S. M., Whitlock H. W., Jr UV shadowing--a new and convenient method for the location of ultraviolet-absorbing species in polyacrylamide gels. Anal Biochem. 1974 May;59(1):162–164. doi: 10.1016/0003-2697(74)90020-7. [DOI] [PubMed] [Google Scholar]
  6. Hutchinson F. The lesions produced by ultraviolet light in DNA containing 5-bromouracil. Q Rev Biophys. 1973 May;6(2):201–246. doi: 10.1017/s0033583500001141. [DOI] [PubMed] [Google Scholar]
  7. Jobe A., Riggs A. D., Bourgeois S. Lac repressor-operator interaction. V. Characterization of super- and pseudo-wild-type repressors. J Mol Biol. 1972 Feb 28;64(1):181–199. doi: 10.1016/0022-2836(72)90328-2. [DOI] [PubMed] [Google Scholar]
  8. Kolchinsky A. M., Mirzabekov A. D., Gilbert W., Li L. Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate. Nucleic Acids Res. 1976 Jan;3(1):11–18. doi: 10.1093/nar/3.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lin S. Y., Riggs A. D. Lac operator analogues: bromodeoxyuridine substitution in the lac operator affects the rate of dissociation of the lac repressor. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2574–2576. doi: 10.1073/pnas.69.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lin S. Y., Riggs A. D. Lac repressor binding to operator analogues: comparison of poly(d(A-T)), poly(d(A-BrU)), and poly(d(A-U)). Biochem Biophys Res Commun. 1971 Dec 17;45(6):1542–1547. doi: 10.1016/0006-291x(71)90195-1. [DOI] [PubMed] [Google Scholar]
  11. Lin S. Y., Riggs A. D. Photochemical attachment of lac repressor to bromodeoxyuridine-substituted lac operator by ultraviolet radiation. Proc Natl Acad Sci U S A. 1974 Mar;71(3):947–951. doi: 10.1073/pnas.71.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lis J. T., Schleif R. Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. Nucleic Acids Res. 1975 Mar;2(3):383–389. doi: 10.1093/nar/2.3.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Platt T., Files J. G., Weber K. Lac repressor. Specific proteolytic destruction of the NH 2 -terminal region and loss of the deoxyribonucleic acid-binding activity. J Biol Chem. 1973 Jan 10;248(1):110–121. [PubMed] [Google Scholar]
  17. Richmond T. J., Steitz T. A. Protein-DNA interaction investigated by binding Escherichia coli lac repressor protein to poly(d(A-U-HgX)). J Mol Biol. 1976 May 5;103(1):25–38. doi: 10.1016/0022-2836(76)90050-4. [DOI] [PubMed] [Google Scholar]
  18. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  19. Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weintraub H. The assembly of newly replicated DNA into chromatin. Cold Spring Harb Symp Quant Biol. 1974;38:247–256. doi: 10.1101/sqb.1974.038.01.028. [DOI] [PubMed] [Google Scholar]
  21. Yansura D. G., Goeddel D. V., Cribbs D. L., Caruthers M. H. Studies of gene control regions. III. Binding of synthetic and modified synthetic lac operator DNAs to lactose repressor. Nucleic Acids Res. 1977 Mar;4(3):723–737. doi: 10.1093/nar/4.3.723. [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