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. 1992 Jan 15;89(2):504–508. doi: 10.1073/pnas.89.2.504

Specific inhibition of transcription by triple helix-forming oligonucleotides.

G Duval-Valentin 1, N T Thuong 1, C Hélène 1
PMCID: PMC48267  PMID: 1731320

Abstract

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

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Selected References

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

  1. Arndt K. M., Chamberlin M. J. RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. J Mol Biol. 1990 May 5;213(1):79–108. doi: 10.1016/S0022-2836(05)80123-8. [DOI] [PubMed] [Google Scholar]
  2. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  3. Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
  4. Cooney M., Czernuszewicz G., Postel E. H., Flint S. J., Hogan M. E. Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science. 1988 Jul 22;241(4864):456–459. doi: 10.1126/science.3293213. [DOI] [PubMed] [Google Scholar]
  5. Drew H. R. Structural specificities of five commonly used DNA nucleases. J Mol Biol. 1984 Jul 15;176(4):535–557. doi: 10.1016/0022-2836(84)90176-1. [DOI] [PubMed] [Google Scholar]
  6. Drew H. R., Travers A. A. Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities. Nucleic Acids Res. 1985 Jun 25;13(12):4445–4467. doi: 10.1093/nar/13.12.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duval-Valentin G., Ehrlich R. Dynamic and structural characterisation of multiple steps during complex formation between E. coli RNA polymerase and the tetR promoter from pSC101. Nucleic Acids Res. 1987 Jan 26;15(2):575–594. doi: 10.1093/nar/15.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duval-Valentin G., Ehrlich R. Far upstream sequences of the bla promoter from TN3 are involved in complexation with E. coli RNA-polymerase. Nucleic Acids Res. 1988 Mar 25;16(5):2031–2044. doi: 10.1093/nar/16.5.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duval-Valentin G., Reiss C. How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter. Mol Microbiol. 1990 Sep;4(9):1465–1475. doi: 10.1111/j.1365-2958.1990.tb02057.x. [DOI] [PubMed] [Google Scholar]
  10. Duval-Valentin G., Schmitt B., Ehrlich R. A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation. Nucleic Acids Res. 1988 Jun 24;16(12):5277–5290. doi: 10.1093/nar/16.12.5277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehrlich R., Larousse A., Jacquet M. A., Marin M., Reiss C. In vitro transcription initiation from three different Escherichia coli promoters. Effect of supercoiling. Eur J Biochem. 1985 Apr 15;148(2):293–298. doi: 10.1111/j.1432-1033.1985.tb08838.x. [DOI] [PubMed] [Google Scholar]
  12. François J. C., Saison-Behmoaras T., Barbier C., Chassignol M., Thuong N. T., Hélène C. Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9702–9706. doi: 10.1073/pnas.86.24.9702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. François J. C., Saison-Behmoaras T., Hélène C. Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies. Nucleic Acids Res. 1988 Dec 23;16(24):11431–11440. doi: 10.1093/nar/16.24.11431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Le Doan T., Perrouault L., Praseuth D., Habhoub N., Decout J. L., Thuong N. T., Lhomme J., Hélène C. Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative. Nucleic Acids Res. 1987 Oct 12;15(19):7749–7760. doi: 10.1093/nar/15.19.7749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee B. L., Murakami A., Blake K. R., Lin S. B., Miller P. S. Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA. Biochemistry. 1988 May 3;27(9):3197–3203. doi: 10.1021/bi00409a011. [DOI] [PubMed] [Google Scholar]
  16. Lomonossoff G. P., Butler P. J., Klug A. Sequence-dependent variation in the conformation of DNA. J Mol Biol. 1981 Jul 15;149(4):745–760. doi: 10.1016/0022-2836(81)90356-9. [DOI] [PubMed] [Google Scholar]
  17. Lyamichev V. I., Mirkin S. M., Frank-Kamenetskii M. D., Cantor C. R. A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs. Nucleic Acids Res. 1988 Mar 25;16(5):2165–2178. doi: 10.1093/nar/16.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maher L. J., 3rd, Dervan P. B., Wold B. J. Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA. Biochemistry. 1990 Sep 18;29(37):8820–8826. doi: 10.1021/bi00489a045. [DOI] [PubMed] [Google Scholar]
  19. Morgan A. R., Wells R. D. Specificity of the three-stranded complex formation between double-stranded DNA and single-stranded RNA containing repeating nucleotide sequences. J Mol Biol. 1968 Oct 14;37(1):63–80. doi: 10.1016/0022-2836(68)90073-9. [DOI] [PubMed] [Google Scholar]
  20. Moser H. E., Dervan P. B. Sequence-specific cleavage of double helical DNA by triple helix formation. Science. 1987 Oct 30;238(4827):645–650. doi: 10.1126/science.3118463. [DOI] [PubMed] [Google Scholar]
  21. Phillips D. R., White R. J., Dean D., Crothers D. M. Monte-Carlo simulation of multisite echinomycin-DNA interactions detected by in vitro transcription analysis. Biochemistry. 1990 May 22;29(20):4812–4819. doi: 10.1021/bi00472a010. [DOI] [PubMed] [Google Scholar]
  22. Praseuth D., Perrouault L., Le Doan T., Chassignol M., Thuong N., Hélène C. Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1349–1353. doi: 10.1073/pnas.85.5.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sun J. S., François J. C., Montenay-Garestier T., Saison-Behmoaras T., Roig V., Thuong N. T., Hélène C. Sequence-specific intercalating agents: intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9198–9202. doi: 10.1073/pnas.86.23.9198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sun J. S., Giovannangeli C., François J. C., Kurfurst R., Montenay-Garestier T., Asseline U., Saison-Behmoaras T., Thuong N. T., Hélène C. Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6023–6027. doi: 10.1073/pnas.88.14.6023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takasugi M., Guendouz A., Chassignol M., Decout J. L., Lhomme J., Thuong N. T., Hélène C. Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5602–5606. doi: 10.1073/pnas.88.13.5602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Waring M. J. DNA modification and cancer. Annu Rev Biochem. 1981;50:159–192. doi: 10.1146/annurev.bi.50.070181.001111. [DOI] [PubMed] [Google Scholar]
  27. Zimmer C., Wähnert U. Nonintercalating DNA-binding ligands: specificity of the interaction and their use as tools in biophysical, biochemical and biological investigations of the genetic material. Prog Biophys Mol Biol. 1986;47(1):31–112. doi: 10.1016/0079-6107(86)90005-2. [DOI] [PubMed] [Google Scholar]

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