Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The kinetic folding pathway of the Tetrahymena ribozyme reveals possible similarities between RNA and protein folding

Abstract

We have probed the nature of the individual kinetic steps in the folding of the Tetrahymena ribozyme by studying the folding kinetics of mutant ribozymes. After rapid formation of the first structural subdomain, a slow step precedes stable formation of the second subdomain. The two central helices of the second subdomain form in an interdependent manner, and this structural subunit therefore also constitutes a kinetic folding unit. The slow folding step includes formation of tertiary interactions in a triple-helical scaffold that orients the two subdomains of the RNA. The rapid and early formation of short range secondary structure, the hierarchical formation of kinetic folding units corresponding to structural subdomains, and the formation of tertiary interactions between subdomains late during the folding process appear to be common features of the folding mechanism for both RNA and proteins.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Pyle, A.M. & Green, J.B. RNA folding. Curr. Opin. Struct. Biol. 5, 303–310 (1995).

    Article  CAS  Google Scholar 

  2. Shen, L.X., Cai, Z. & Tinoco, I., jr. RNA structure at high resolution. FASEB J. 9, 1023–1033 (1995).

    Article  CAS  Google Scholar 

  3. Banerjee, A.R. & Turner, D.H. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry 34, 6504–6512 (1995).

    Article  CAS  Google Scholar 

  4. Bassi, G.S., MØIIegaard, N.-E., Murchie, A.I.H., von Kitzing, E. & Lilley, D.M.J. Ionic interactions and the global conformations of the hammerhead ribozyme. Nature Struct. Biol. 2, 45–55 (1995).

    Article  CAS  Google Scholar 

  5. Celander, D.W. & Cech, T.R. Visualizing the higher order folding of a catalytic RNA molecule. Science 251, 401–407 (1991).

    Article  CAS  Google Scholar 

  6. Herschlag, D. RNA chaperones and the RNA folding problem. J. Biol. Chem. 270, 20871–20874 (1995).

    Article  CAS  Google Scholar 

  7. Jaeger, L., Westhof, E. & Michel, F. Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. The active form of the sunY ribozyme is stabilized by multiple interactions with 3′ terminal intron components. J. Mol. Biol. 234, 331–346 (1993).

    Article  CAS  Google Scholar 

  8. Laggerbauer, B., Murphy, F.L. & Cech, T.R. Two major tertiary folding transitions of the Tetrahymena catalytic RNA. EMBO J. 13, 2669–2676 (1994).

    Article  CAS  Google Scholar 

  9. Ma, C.K. et al. Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation. Mol. Microbiol. 14, 1033–1047 (1994).

    Article  CAS  Google Scholar 

  10. Pan, T. Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 34, 902–909 (1995).

    Article  CAS  Google Scholar 

  11. Weeks, K.M. & Cech, T.R. Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5′ splice site domain. Cell 82, 221–230 (1995).

    Article  CAS  Google Scholar 

  12. Weeks, K.M. & Cech, T.R. Assembly of a ribonucleoprotein catalyst by tertiary structure capture. Science 271, 345–348 (1996).

    Article  CAS  Google Scholar 

  13. Zarrinkar, P.P. & Williamson, J.R. Kinetic intermediates in RNA folding. Science 265, 918–924 (1994).

    Article  CAS  Google Scholar 

  14. Zarrinkar, P.P. & Williamson, J.R. The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. 24, 854–858 (1996).

    Article  CAS  Google Scholar 

  15. Cech, T.R. Structure and mechanism of the large catalytic RNAs: group I and group II introns and ribonuclease P. in The RNA World (eds Gesteland, R.F. & Atkins, J.F.) 239–269 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1993).

    Google Scholar 

  16. Cech, T.R., Damberger, S.H. & Gutell, R.R. Representation of the secondary and tertiary structure of group I introns. Nature Struct. Biol. 1, 273–280 (1994).

    Article  CAS  Google Scholar 

  17. Michel, F. & Westhof, E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610 (1990).

    Article  CAS  Google Scholar 

  18. Doudna, J.A. & Cech, T.R. Self-assembly of a group I intron active site from its component tertiary structural domains. RNA 1, 36–45 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Murphy, F.L. & Cech, T.R. An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme. Biochemistry 32, 5291–5300 (1993).

    Article  CAS  Google Scholar 

  20. Nakamura, T.M., Wang, Y.-H., Zaug, A.J., Griffith, J.D. & Cech, T.R. Relative orientation of RNA helices in a group I ribozyme determined by helix extension electron microscopy. EMBO J. 14, 4849–4859 (1995).

    Article  CAS  Google Scholar 

  21. Wang, Y.-H., Murphy, F.L., Cech, T.R. & Griffith, J.D. Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy. J. Mol. Biol. 236, 64–71 (1994).

    Article  CAS  Google Scholar 

  22. Burke, J.M. et al. Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor. Cell 45, 167–176 (1986).

    Article  CAS  Google Scholar 

  23. Williamson, C.L., Tierney, W.M., Kerker, B.J. & Burke, J.M. Site-directed mutagenesis of core sequence elements 9R′, 9L, 9R and 2 in self-splicing Tetrahymena pre-rRNA. J. Biol. Chem. 262, 14672–14682 (1987).

    CAS  PubMed  Google Scholar 

  24. Michel, F., Ellington, A.D., Couture, S. & Szostak, J.W. Phylogenetic and genetic evidence for base-triples in the catalytic domain of group I introns. Nature 347, 578–580 (1990).

    Article  CAS  Google Scholar 

  25. Green, R. & Szostak, J.W. In vitro genetic analysis of the hinge region between helical elements P5-P4-P6 and P7-P3-P8 in the sunY group I self-splicing intron. J. Mol. Biol. 235, 140–155 (1994).

    Article  CAS  Google Scholar 

  26. Chastain, M. & Tinoco, I., jr. Nucleoside triples from the group I intron. Biochemistry 32, 14220–14228 (1993).

    Article  CAS  Google Scholar 

  27. Herschlag, D. & Cech, T.R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry 29, 10159–10171 (1990).

    Article  CAS  Google Scholar 

  28. McConnell, T.S., Cech, T.R. & Herschlag, D. Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding. Proc. Natl. Acad. Sci. USA 90, 8362–8366 (1993).

    Article  CAS  Google Scholar 

  29. Murphy, F.L. & Cech, T.R. GAAA tetraloop and conserved bulge stabilize tertiary structure of a group I intron domain. J. Mol. Biol. 236, 49–63 (1994).

    Article  CAS  Google Scholar 

  30. Mohr, G., Zhang, A., Gianelos, J.A., Belfort, M. & Lambowitz, A.M. The Neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core. Cell 69, 483–494 (1992).

    Article  CAS  Google Scholar 

  31. Christensen, H. & Pain, R.H. The contribution of the molten globule model. in Mechanisms of Protein Folding (ed. Pain, R.H.) 55–79 (Oxford University Press, New York, 1994).

    Google Scholar 

  32. Lynch, D.C. & Schimmel, P.R. Cooperative binding of magnesium to transfer ribonucleic acid studied by a fluorescent probe. Biochemistry 13, 1841–1852 (1974).

    Article  CAS  Google Scholar 

  33. Stein, A. & Crothers, D.M. Conformational changes of transfer RNA. The role of magnesium(II). Biochemistry 15, 160–168 (1976).

    Article  CAS  Google Scholar 

  34. Westhof, E. & Michel, F. Some tertiary motifs of RNA foldings. in Structural Tools for the Analysis of Protein-Nucleic Acid Complexes (eds Lilley, D.J.M., Heumann, H. & Suck, D.) 255–267 (Birkhäuser Verlag, Basel, 1992).

    Google Scholar 

  35. Bai, Y., Sosnick, T., Mayne, L. & Englander, S.W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).

    Article  CAS  Google Scholar 

  36. Dobson, C.M. Finding the right fold. Nature Struct. Biol. 2, 513–517 (1995).

    Article  CAS  Google Scholar 

  37. Dobson, C.M., Evans, P.A. & Radford, S.E. Understanding how proteins fold: the lysozyme story so far. TIBS 19, 31–37 (1994).

    CAS  PubMed  Google Scholar 

  38. Jennings, P.A. & Wright, P.E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–896 (1993).

    Article  CAS  Google Scholar 

  39. López-Hernandez, E. & Serrano, L. Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI–2. Folding & Design 1, 43–55 (1996).

    Article  Google Scholar 

  40. Serrano, L., Matouschek, A. & Fersht, A.R. The folding of an enzyme VI. The folding pathway of barnase: comparison with theoretical models. J. Mol. Biol. 224, 847–859 (1992).

    Article  CAS  Google Scholar 

  41. Wu, L.C., Peng, Z.-y. & Kim, P.S. Bipartite structure of the α-lactalbumin molten globule. Nature Struct. Biol. 2, 281–286 (1995).

    Article  CAS  Google Scholar 

  42. Loh, S.N., Kay, M.S. & Baldwin, R.L. Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway. Proc. Natl. Acad. Sci. USA 92, 5446–5450 (1995).

    Article  CAS  Google Scholar 

  43. Higuchi, R. Recombinant PCR. in PCR Protocols: A Guide to Methods and Applications (eds Innis, M.A. et al.) 177–183 (Academic Press Inc., New York, 1990).

    Google Scholar 

  44. Zaug, A.J., Grosshans, C.A. & Cech, T.R. Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry 27, 8924–8931 (1988).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zarrinkar, P., Williamson, J. The kinetic folding pathway of the Tetrahymena ribozyme reveals possible similarities between RNA and protein folding. Nat Struct Mol Biol 3, 432–438 (1996). https://doi.org/10.1038/nsb0596-432

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0596-432

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing