Biochemistry (Moscow)

, Volume 75, Issue 4, pp 405–411 | Cite as

Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA

  • N. E. Shirokikh
  • S. Ch. Agalarov
  • A. S. SpirinEmail author
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The 5′-untranslated sequence of tobacco mosaic virus RNA — the so-called omega leader — exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson-Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson-Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson-Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson-Crick type.

Key words

omega leader of TMV RNA regular (CAA)n polyribonucleotide RNA triple helix chemical modification of RNA enzymatic cleavage of RNA primer extension inhibition diethyl pyrocarbonate dimethyl sulfate RNase A RNase V1 



avian myeloblastosis virus


complementary DNA






4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid


tobacco mosaic virus




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  1. 1.
    Sleat, D. E., Gallie, D. R., Jefferson, R. A., Bevan, M. W., Turner, P. C., and Wilson, T. M. (1987) Gene, 60, 217–225.CrossRefPubMedGoogle Scholar
  2. 2.
    Gallie, D. R. (2002) Nucleic Acids Res., 30, 3401–3411.CrossRefPubMedGoogle Scholar
  3. 3.
    Alekhina, O. M., Vassilenko, K. S., and Spirin, A. S. (2007) Nucleic Acids Res., 35, 6547–6559.CrossRefPubMedGoogle Scholar
  4. 4.
    Zeyenko, V. V., Ryabova, L. A., Gallie, D. R., and Spirin, A. S. (1994) FEBS Lett., 354, 271–273.CrossRefPubMedGoogle Scholar
  5. 5.
    Saejung, W., Fujiyama, K., Takasaki, T., Ito, M., Hori, K., Malasit, P., Watanabe, Y., Kurane, I., and Seki, T. (2007) Vaccine, 25, 6646–6654.CrossRefPubMedGoogle Scholar
  6. 6.
    Schmitz, J., Prufer, D., Rohde, W., and Tacke, E. (1996) Nucleic Acids Res., 24, 257–263.CrossRefPubMedGoogle Scholar
  7. 7.
    Gallie, D. R., Walbot, V., and Hershey, J. W. (1988) Nucleic Acids Res., 16, 8675–8694.CrossRefPubMedGoogle Scholar
  8. 8.
    Gallie, D. R., Sleat, D. E., Watts, J. W., Turner, P. C., and Wilson, T. M. (1988) Nucleic Acids Res., 16, 883–893.CrossRefPubMedGoogle Scholar
  9. 9.
    Tzareva, N. V., Makhno, V. I., and Boni, I. V. (1994) FEBS Lett., 337, 189–194.CrossRefPubMedGoogle Scholar
  10. 10.
    Kopeina, G. S., Afonina, Z. A., Gromova, K. V., Shirokov, V. A., Vasiliev, V. D., and Spirin, A. S. (2008) Nucleic Acids Res., 36, 2476–2488.CrossRefPubMedGoogle Scholar
  11. 11.
    Kukla, B. A., Guilley, H. A., Jonard, G. X., Richards, K. E., and Mundry, K. W. (1979) Eur. J. Biochem., 98, 61–66.CrossRefPubMedGoogle Scholar
  12. 12.
    Gallie, D. R., and Walbot, V. (1992) Nucleic Acids Res., 20, 4631–4638.CrossRefPubMedGoogle Scholar
  13. 13.
    Mundry, K. W., Watkins, P. A., Ashfield, T., Plaskitt, K. A., Eisele-Walter, S., and Wilson, T. M. (1991) J. Gen. Virol., 72(Pt. 4), 769–777.CrossRefPubMedGoogle Scholar
  14. 14.
    Saenger, W. (1984) in Principles of Nucleic Acid Structure, Springer, New York.Google Scholar
  15. 15.
    Gudkov, A. T., Ozerova, M. V., Shiryaev, V. M., and Spirin, A. S. (2005) Biotechnol. Bioeng., 91, 468–473.CrossRefPubMedGoogle Scholar
  16. 16.
    Kovtun, A. A., Shirokikh, N. E., Gudkov, A. T., and Spirin, A. S. (2007) Biochem. Biophys. Res. Commun., 358, 368–372.CrossRefPubMedGoogle Scholar
  17. 17.
    Efimov, A. V., and Spirin, A. S. (2009) Biochem. Biophys. Res. Commun., 388, 127–130.CrossRefPubMedGoogle Scholar
  18. 18.
    Huntzinger, E., Possedko, M., Winter, F., Moine, H., Ehresmann, C., Romby, P., (2008) in Handbook of RNA Biochemistry (Hartmann, R. K., Bindereif, A., Schon, A., and Westhof, E., eds.) Wiley-VCH Verlag, Weinheim, pp. 151–171.Google Scholar
  19. 19.
    Merryman, C., and Noller, H. F. (1998) in RNA:Protein Interactions, A Practical Approach (Smith, C. W. J., ed.) Oxford University Press, New York, pp. 237–253.Google Scholar
  20. 20.
    Gurevich, V. V. (1996) Meth. Enzymol., 275, 382–397.CrossRefPubMedGoogle Scholar
  21. 21.
    Gurevich, V. V., Pokrovskaya, I. D., Obukhova, T. A., and Zozulya, S. A. (1991) Anal. Biochem., 195, 207–213.CrossRefPubMedGoogle Scholar
  22. 22.
    Shirokikh, N. E., and Spirin, A. S. (2008) Proc. Natl. Acad. Sci. USA, 105, 10738–10743.CrossRefPubMedGoogle Scholar
  23. 23.
    Shirokikh, N. E., Alkalaeva, E. Z., Vassilenko, K. S., Afonina, Z. A., Alekhina, O. M., Kisselev, L. L., and Spirin, A. S. (2009) Nucleic Acids Res., doi: 10.1093/nar/gkp1025.Google Scholar
  24. 24.
    Fekete, R. A., Miller, M. J., and Chattoraj, D. K. (2003) Biotechniques, 35, 90–98.PubMedGoogle Scholar
  25. 25.
    Yindeeyoungyeon, W., and Schell, M. A. (2000) Biotechniques, 29, 1034–1041.PubMedGoogle Scholar
  26. 26.
    Gould, P. S., Bird, H., and Easton, A. J. (2005) Biotechniques, 38, 397–400.CrossRefPubMedGoogle Scholar
  27. 27.
    Peattie, D. A., and Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA, 77, 4679–4682.CrossRefPubMedGoogle Scholar
  28. 28.
    Mandiyan, V., and Boublik, M. (1990) Nucleic Acids Res., 18, 7055–7062.CrossRefPubMedGoogle Scholar
  29. 29.
    Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezancon, A., Ebel, J. P., Ehresmann, B., and Ehresmann, C. (1987) J. Mol. Biol., 198, 91–107.CrossRefPubMedGoogle Scholar
  30. 30.
    Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA, 74, 560–564.CrossRefPubMedGoogle Scholar
  31. 31.
    Leonard, N. J., McDonald, J. J., Henderson, R. E., and Reichmann, M. E. (1971) Biochemistry, 10, 3335–3342.CrossRefPubMedGoogle Scholar
  32. 32.
    Weeks, K. M., and Crothers, D. M. (1993) Science, 261, 1574–1577.CrossRefPubMedGoogle Scholar
  33. 33.
    Lockard, R. E., and Kumar, A. (1981) Nucleic Acids Res., 9, 5125–5140.CrossRefPubMedGoogle Scholar
  34. 34.
    Kop, J., Kopylov, A. M., Magrum, L., Siegel, R., Gupta, R., Woese, C. R., and Noller, H. F. (1984) J. Biol. Chem., 259, 15287–15293.PubMedGoogle Scholar
  35. 35.
    Hartshorne, T., and Agabian, N. (1994) Nucleic Acids Res., 22, 3354–3364.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • N. E. Shirokikh
    • 1
  • S. Ch. Agalarov
    • 1
  • A. S. Spirin
    • 1
    Email author
  1. 1.Laboratory of Mechanisms of Protein Biosynthesis, Institute of Protein ResearchRussian Academy of SciencesPushchino, Moscow RegionRussia

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