Skip to main content
SpringerLink
Log in

The dynamic NMR structure of the TΨC-loop: Implications for the specificity of tRNA methylation

  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependentmethylation of uridine-54 in the TΨC-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried andthe question arises as to how RUMT gains access to the methylation site. A 17-mer RNAhairpin consisting of nucleotides 49–65 of the TΨ-loop is a substrate for RUMT.Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD)methods were used to determine the solution structure of the 17-mer T-arm fragment. Theloop of the hairpin exhibits enhanced flexibility which renders the conventional NMR averagestructure less useful compared to the more commonly found situation where a molecule existsin predominantly one major conformation. However, when resorting to softer refinementmethods such as MD with time-averaged restraints, the conflicting restraints in the loop canbe satisfied much better. The dynamic structure of the T-arm is represented as an ensembleof 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the TΨ-loop in solution in conjunction with extensive binding studies of RUMT with the TΨC-loop and tRNA suggest that the specificity of the RUMT/tRNA recognition is associated withtRNA tertiary structure elements. For the methylation, RUMT would simply have to breakthe tertiary interactions between the D- and T-loops, leading to a melting of the T-armstructure and making U54 available for methylation.

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

References

  • Amano, M. and Kawakami, M. (1992) Eur. J. Biochem., 210, 671–681.

    Google Scholar 

  • Bax, A. and Davis, D.G. (1985) J. Magn. Reson., 65, 355–372.

    Google Scholar 

  • Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., Di Nola, A. and Haak, J.R. (1984) J. Chem. Phys., 81, 3684–3690.

    Google Scholar 

  • Bonvin, A.M.J.J. and Brünger, A.T. (1995) J. Mol. Biol., 250, 80–93.

    Google Scholar 

  • Bonvin, A.M.J.J. and Brünger, A.T. (1996) J. Biomol. NMR, 7, 72–76.

    Google Scholar 

  • Fennen, J., Torda, A.E. and van Gunsteren, W.F. (1995) J. Biomol. NMR, 6, 163–170.

    Google Scholar 

  • Gallo, K., Huang, C., Ferrin, T.F. and Langridge, R. (1985, 1989) Molecular Interactive Display and Simulation (MidasPlus), University of California, San Francisco, CA, U.S.A.

    Google Scholar 

  • González, C., Stec, W., Reynolds, M. and James, T.L. (1995) Biochemistry, 34, 4969–4982.

    Google Scholar 

  • Gu, X., Ivanetich, K.M. and Santi, D.V. (1996) Biochemistry, 35, 11652–11659.

    Google Scholar 

  • Hall, K.B., Sampson, J.R., Uhlenbeck, O.C. and Redfield, A.G. (1989) Biochemistry, 28, 5791–5801.

    Google Scholar 

  • Heerschap, A., Haasnoot, C.A.G. and Hilbers, C.W. (1983a) Nucleic Acids Res., 11, 4483–4499.

    Google Scholar 

  • Heerschap, A., Haasnoot, C.A.G. and Hilbers, C.W. (1983b) Nucleic Acids Res., 11, 4501–4520.

    Google Scholar 

  • Heus, H.A. and Pardi, A. (1991) J. Am. Chem. Soc., 113, 4360–4361.

    Google Scholar 

  • Holbrook, S.R., Sussman, J.L., Warrant, R.W. and Kim, S.-H. (1978) J. Mol. Biol., 123, 631–660.

    Google Scholar 

  • Hyde, E.I. and Reid, B.R. (1985) Biochemistry, 24, 4307–4314.

    Google Scholar 

  • Jaeger, J.A. and Tinoco Jr., I. (1993) Biochemistry, 32, 12522–12530.

    Google Scholar 

  • James, T.L. (1991) Curr. Opin. Struct. Biol., 1, 1042–1053.

    Google Scholar 

  • Kealey, J.T., Gu, X. and Santi, D.V. (1994) Biochimie, 76, 1133–1142.

    Google Scholar 

  • Keepers, J.W. and James, T.L. (1984) J. Magn. Reson., 57, 404–426.

    Google Scholar 

  • Kemmink, J. and Scheek, R.M. (1995) J. Biomol. NMR, 6, 33–40.

    Google Scholar 

  • Kneller, D.G., Sparky, NMR display and processing program, University of California, San Francisco, CA, U.S.A., © 1992.

    Google Scholar 

  • Liu, H., Borgias, B., Kumar, A. and James, T.L. (1990, 1994) MARDIGRAS, University of California, San Francisco, CA, U.S.A.

    Google Scholar 

  • Liu, H., Spielmann, H.P., Ulyanov, N.B., Wemmer, D.E. and James, T.L. (1995) J. Biomol. NMR, 6, 390–402.

    Google Scholar 

  • Louise-May, S., Auffinger, P. and Westhof, E. (1996) In Biological Structure and Dynamics(Eds., Sarma, R.H. and Sarma, M.H.), Vol. 2, Adenine Press, Schenectady, NY, U.S.A., pp. 73–91.

    Google Scholar 

  • Milligan, J.F., Groebe, D.R., Wilherell, G.W. and Uhlenbeck, O.C. (1987) Nucleic Acids Res., 15, 8783–8798.

    Google Scholar 

  • Nanzer, A.P., van Gunsteren, W.F. and Torda, A.E. (1995) J. Biomol. NMR, 6, 313–320.

    Google Scholar 

  • Pan, T., Long, D.M. and Uhlenbeck, O. (1993) In The RNA World (Eds., Gesteland, R.F. and Atkins, J.F.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A., pp. 271–302.

    Google Scholar 

  • Pearlman, D.A. and Kollman, P.A. (1991) J. Mol. Biol., 220, 429–457.

    Google Scholar 

  • Pearlman, D.A. (1994) J. Biomol. NMR, 4, 1–16.

    Google Scholar 

  • Pearlman, D.A., Case, D.A., Caldwell, J.C., Ross, W.S., Cheatham III, T.E., Ferguson, D.N., Seibel, G.L., Singh, U.C., Weiner, P.K. and Kollman, P.A. (1995) AMBER v. 4.1, University of San Francisco, San Francisco, CA, U.S.A.

    Google Scholar 

  • Puglisi, J.D. and Tinoco, I.J. (1989) Methods Enzymol., 180, 304–325.

    Google Scholar 

  • Quigley, G.J. and Rich, A. (1976) Science, 194, 796–806.

    Google Scholar 

  • Romby, P., Acabon, P., Westhof, E., Ehresmann, C., Ebel, J.-P., Ehresmann, B. and Giege, R. (1987) J. Biomol. Struct. Dyn., 5, 669–687.

    Google Scholar 

  • Roy, S. and Redfield, A.G. (1983) Biochemistry, 22, 1386–1390.

    Google Scholar 

  • Ryckaert, J.P., Cicotti, G. and Berendsen, H.J.C. (1977) J. Comput. Phys., 23, 327–341.

    Google Scholar 

  • Schmitz, U., Kumar, A. and James, T.L. (1992) J. Am. Chem. Soc., 114, 10564–10566.

    Google Scholar 

  • Schmitz, U. and James, T.L. (1993) In Structural Biology: The State of the Art(Eds., Sarma, R.H. and Sarma, M.H.), Vol. 2, Adenine Press, Schenectady, NY, U.S.A., pp. 251–272.

    Google Scholar 

  • Schmitz, U., Ulyanov, N.B., Kumar, A. and James, T.L. (1993) J. Mol. Biol., 234, 373–389.

    Google Scholar 

  • Schmitz, U. and James, T.L. (1995) Methods Enzymol., 261, 1–43.

    Google Scholar 

  • Schmitz, U., González, C., Liu, H., Ulyanov, N.B., Blocker, F. and James, T.L. (1996) In Biological Structure and Dynamics(Eds., Sarma, R.H. and Sarma, M.H.), Vol. 2, Adenine Press, Schenectady, NY, U.S.A., pp. 165–189.

    Google Scholar 

  • Seibel, G.L., Singh, U.C. and Kollman, P.A. (1985) Proc. Natl. Acad. Sci. USA, 82, 6537–6540.

    Google Scholar 

  • Shen, L.X., Cai, Z. and Tinoco, I. (1995) FASEB J., 9, 1023–1033.

    Google Scholar 

  • Simmerling, C., Elber, R. and Zhang, J. (1995) In Modeling of Biomolecular Structures and Mechanisms(Ed., Pullman, A.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 241–265.

    Google Scholar 

  • Smallcombe, S.H. (1993) J. Am. Chem. Soc., 115, 4776–4785.

    Google Scholar 

  • States, D.J., Haberkorn, R.A. and Ruben, D.J. (1982) J. Magn. Reson., 48, 286–292.

    Google Scholar 

  • Torda, A.E., Scheek, R.M. and van Gunsteren, W.F. (1990) J. Mol. Biol., 214, 223–235.

    Google Scholar 

  • Torda, A.E., Brunne, R.M., Huber, T., Kessler, H. and van Gunsteren, W.F. (1993) J. Biomol. NMR, 3, 55–66.

    Google Scholar 

  • Ulyanov, N.B., Schmitz, U., Kumar, A. and James, T.L. (1995) Biophys. J., 68, 13–24.

    Google Scholar 

  • Varani, G. and Tinoco, I.J. (1991) Q. Rev. Biophys., 24, 479–532.

    Google Scholar 

  • Wijmenga, S.S., Mooren, M.M.W. and Hilbers, C.W. (1993) In NMR of Macromolecules(Ed., Roberts, G.C.K.), Vol. 134, Oxford University Press, New York, NY, U.S.A., pp. 217–288.

    Google Scholar 

  • Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York, NY, U.S.A.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0446, U.S.A

    Letitia J. Yao, Thomas L. James, James T. Kealey, Daniel V. Santi & Uli Schmitz

Authors
  1. Letitia J. Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas L. James
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James T. Kealey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel V. Santi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Uli Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yao, L.J., James, T.L., Kealey, J.T. et al. The dynamic NMR structure of the TΨC-loop: Implications for the specificity of tRNA methylation. J Biomol NMR 9, 229–244 (1997). https://doi.org/10.1023/A:1018618606857

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1018618606857

Search

Navigation