Skip to main content
SpringerLink
Log in

The environment of tRNA 3′-terminus in 80S ribosomal A and P sites

  • Published:
Russian Journal of Bioorganic Chemistry Aims and scope Submit manuscript

Abstract

The environment of tRNA 3′-terminus in the 80S ribosomal A and P sites was studied with a tRNAAsp analogue that bears a 4-thiouridine residue (s4U) attached to the 3′-terminal adenosine. The tRNAAsp analogue was obtained by in vitro T7 transcription followed by crosslinking with [32P]ps4Up and removal of the 3′-terminal phosphate. It was shown that the presence of the additional nucleotide at the 3′-end does not to hinder the codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in crosslinking of the analogue exclusively to 28S rRNA. The crosslinking was completely dependent on the presence of s4U in the tRNA analogue. Using hydrolysis of the crosslinked 28S rRNA with RNase H in the presence of deoxyoligomers complementary to various rRNA sequences, we determined that the crosslinking occurred in fragment 4302–4540 of the 28S rRNA. This fragment is evolutionarily conservative and belongs to domain V that is involved in the formation of the peptidyl transferase site in prokaryotic ribosomes. The use of reverse transcription allowed the determination of the tRNA analogue crosslinking in the P site to nucleotides U4461 and U4502, and the analogue in the A site, to nucleotides U4469 and C4507. In addition, nucleotide C4462 was crosslinked to both P site and A site-bound tRNA analogue. An analysis of the results demonstrates that environments of the tRNA 3′-termini are closely similar in both prokaryotic and eukaryotic ribosomes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

PTC:

peptidyl transferase site

References

  1. Yusupova, G.Zh., Yusupov, M.M., Cate, J.H.D., and Noller, H.F., Cell, 2001, vol. 106, pp. 233–241.

    Article  CAS  PubMed  Google Scholar 

  2. Bashan, A., Agmon, I., Zarivach, R., Schluenzen, F., Harms, J., Berisio, R., Bartels, H., Franceschi, F., Auerbach, T., Hansen, H.A.S., Kossoy, E., Kessler, M., and Yonath, A., Mol. Cell, 2003, vol. 11, pp. 91–102.

    Article  CAS  PubMed  Google Scholar 

  3. Steitz, T.A., FEBS Lett., 2005, vol. 579, pp. 955–958.

    Article  CAS  PubMed  Google Scholar 

  4. Graifer, D.M., Karpova, G.G., and Knorre, D.G., Biokhimiya (Moscow), 2001, vol. 66, pp. 725–744.

    Article  Google Scholar 

  5. Graifer, D.M. and Karpova, G.G., Mol. Biol. (Moscow), 2001, vol. 35, pp. 584–596.

    Article  CAS  Google Scholar 

  6. Smolenskaya, I.A., Bulygin, K.N., Graifer, D.M., Ivanov, A.V., Ven’yaminova, A.G., Repkova, M.N., and Karpova, G.G., Mol. Biol. (Moscow), 1998, vol. 32, pp. 233–241.

    Google Scholar 

  7. Demeshkina, N.A., Laletina, E.S., Meshchaninova, M.I., Repkova, M.N., Ven’yaminova, A.G., Graifer, D.M., and Karpova, G.G., Mol. Biol. (Moscow), 2003, vol. 37, pp. 147–155.

    CAS  Google Scholar 

  8. Graifer, D., Molotkov, M., Styazhkina, V., Demeshkina, N., Bulygin, K., Eremina, A., Ivanov, A., Laletina, E., Ven’yaminova, A., and Karpova, G., Nucleic Acids Res., 2004, vol. 32, pp. 3282–3293.

    Article  CAS  PubMed  Google Scholar 

  9. Molotkov, M.V., Graifer, D.M., Meshchaninova, M.I., Repkova, M.N., Ven’yaminova, A.G., and Karpova, G.G., Mol. Biol. (Moscow), 2004, vol. 38, pp. 493–500.

    CAS  Google Scholar 

  10. Molotkov, M.V., Graifer, D.M., Eremina, A.V., Ivanov, A.V., Laletina, E.S., Repkova, M.N., Ven’yaminova, A.G., and Karpova, G.G., Mol. Biol. (Moscow), 2004, vol. 38, pp. 1033–1040.

    CAS  Google Scholar 

  11. Molotkov, M., Graifer, D., Demeshkina, N., Repkova, M., Ven’yaminova, A., and Karpova, G., RNA Biology, 2005, vol. 2, pp. 63–69.

    CAS  PubMed  Google Scholar 

  12. Favre, A., Saintome, C., Fourrey, J.-L., Clivio, P., and Laugaa, P., Photochem. Photobiol., 1997, vol. 42, pp. 109–124.

    Google Scholar 

  13. Sergiev, P.V., Dontsova, O.A., and Bogdanov, A.A., Mol. Biol. (Moscow), 2001, vol. 35, pp. 559–583.

    Article  CAS  Google Scholar 

  14. Graifer, D.M., Juzumiene, D.I., Karpova, G.G., and Wollenzien, P., Biochemistry, 1994, vol. 33, pp. 6201–6206.

    Article  CAS  PubMed  Google Scholar 

  15. Graifer, D.M., Juzumiene, D.I., Wollenzien, P., and Karpova, G.G., Biochemistry, 1994, vol. 33, pp. 3878–3884.

    Article  CAS  PubMed  Google Scholar 

  16. Bulygin, K., Chavatte, L., Frolova, L., Karpova, G., and Favre, A., Biochemistry, 2005, vol. 44, pp. 2153–2162.

    Article  CAS  PubMed  Google Scholar 

  17. Chavatte, L., Frolova, L.Yu., Kisselev, L.L., and Favre, A., Eur. J. Biochem., 2001, vol. 268, pp. 2896–2904.

    Article  CAS  PubMed  Google Scholar 

  18. Chavatte, L., Seit-Nebi, A., Dubovaya, V., and Favre, A., EMBO J., 2002, vol. 21, pp. 5302–5311.

    Article  CAS  PubMed  Google Scholar 

  19. Moor, N.A., Ankilova, V.N., Lavrik, O.I., and Favre, A., Biochim. Biophys. Acta, 2001, vol. 1518, pp. 226–236.

    CAS  PubMed  Google Scholar 

  20. Graifer, D.M., Nekhai, S.Yu., Mundus, D.A., Fedorova, O.S., and Karpova, G.G., Biochim. Biophys. Acta, 1992, vol. 1171, pp. 56–64.

    CAS  PubMed  Google Scholar 

  21. Selmer, M. Dunham, C.M., Murphy IV, F.M., Weixlbaumer, A., Petry, S., Kelley, A.C., Weir, J.R., and Ramakrishnan, V., Science, 2006, vol. 313, pp. 1935–1942.

    Article  CAS  PubMed  Google Scholar 

  22. Yusupov, M.M., Yusupova, G.Zh., Baucom, A., Lieberman, K., Earnest, T., Cate, J.H.D., and Noller, H.F., Science, 2001, vol. 292, pp. 883–896.

    Article  CAS  PubMed  Google Scholar 

  23. Gerbi, S.A., in Ribosomal RNA: Structure, Evolution, Processing, and Function in Protein Biosynthesis, Zimmermann, R.A. and Dahlberg, A.E., Eds., Boca Raton; NY: London: CRC, 1996, pp. 71–87.

    Google Scholar 

  24. Ven’yaminova, A.G., Gorn, V.V., Zenkova, M.A., Komarova, N.I., and Repkova, M.N., Bioorg. Khim., 1990, vol. 16, pp. 941–950.

    Google Scholar 

  25. Matasova, N.B., Myltseva, S.V., Zenkova, M.A., Graifer, D.M., Vladimirov, S.N., and Karpova, G.G., Anal. Biochem., 1991, vol. 198, pp. 219–223.

    Article  CAS  PubMed  Google Scholar 

  26. Smolenskaya, I.A., Graifer, D.M., Ivanov, A.V., Vladimirov, S.N., Ven’yaminova, A.G., Repkova, M.N., Shtal, I., and Karpova, G.G., Mol. Biol. (Moscow), 1997, vol. 31, pp. 144–151.

    Google Scholar 

  27. Malygin, A.A., Graifer, D.M., Bulygin, K.N., Zenkova, M.A., Yamkovoy, V.I., Stahl, J., and Karpova, G.G., Eur. J. Biochem., 1994, vol. 226, pp. 715–723.

    Article  CAS  PubMed  Google Scholar 

  28. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.

    Article  CAS  PubMed  Google Scholar 

  29. Graifer, D.M., Malygin, A.A., Matasova, N.B., Mundus, D.A., Zenkova, M.A., and Karpova, G.G., Biochim. Biophys. Acta, 1997, vol. 1350, pp. 335–344.

    CAS  PubMed  Google Scholar 

  30. Wollenzien, P., Methods Enzymol., 1988, vol. 164, pp. 319–329.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, 630090, Russia

    K. N. Bulygin, D. M. Graifer, A. G. Ven’yaminova & G. G. Karpova

  2. Institut Jacques Monod, UMR 7592 CNRS-Universites Paris 7 — Paris 6, 2 place Jussieu Tour 43, 75251, Paris Cedex 05, France

    S. Baouz-Drahy & A. Favre

Authors
  1. K. N. Bulygin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Baouz-Drahy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Favre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. M. Graifer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. G. Ven’yaminova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G. G. Karpova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. G. Karpova.

Additional information

Original Russian Text © K.N. Bulygin, S. Baouz-Drahy, A. Favre, D.M. Graifer, A.G. Ven’yaminova, G.G. Karpova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 1, pp. 96–106.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bulygin, K.N., Baouz-Drahy, S., Favre, A. et al. The environment of tRNA 3′-terminus in 80S ribosomal A and P sites. Russ J Bioorg Chem 34, 87–96 (2008). https://doi.org/10.1134/S1068162008010123

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1068162008010123

Key words

Search

Navigation