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.

Multiple native states reveal persistent ruggedness of an RNA folding landscape

Nature volume 463, pages 681–684 (2010)Cite this article

Subjects

Abstract

According to the ‘thermodynamic hypothesis’, the sequence of a biological macromolecule defines its folded, active (or ‘native’) structure as a global energy minimum in the folding landscape1,2. However, the enormous complexity of folding landscapes of large macromolecules raises the question of whether there is in fact a unique global minimum corresponding to a unique native conformation or whether there are deep local minima corresponding to alternative active conformations3. The folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation4,5. Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert6,7,8. Here we demonstrate, using single-molecule experiments, that an RNA enzyme folds into multiple distinct native states that interconvert on a timescale much longer than that of catalysis. These data demonstrate that severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Docking and cleavage of the oligonucleotide substrate by the Tetrahymena ribozyme, observed using single-molecule FRET.
Figure 2: Distribution of docking behaviours of individual ribozyme molecules.
Figure 3: Catalytic activities of molecules from different parts of docking distribution are the same.
Figure 4: Interconversion of docking behaviours.

References

  1. Anfinsen, C. B. Principles that govern the folding of protein chains. Science 181, 223–230 (1973)

    Article  ADS  CAS  Google Scholar 

  2. Bryngelson, J. D., Onuchic, J. N., Socci, N. D. & Wolynes, P. G. Funnels, pathways and the energy landscape of protein folding: a synthesis. Proteins Struct. Funct. Genet. 21, 167–195 (1995)

    Article  CAS  Google Scholar 

  3. James, L. C. & Tawfik, D. S. Conformational diversity and protein evolution - a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 (2003)

    Article  CAS  Google Scholar 

  4. Zwanzig, R. Two-state models of protein folding kinetics. Proc. Natl Acad. Sci. USA 94, 148–150 (1997)

    Article  ADS  CAS  Google Scholar 

  5. Dill, K. A. & Chan, H. S. From Levinthal to pathways to funnels. Nature Struct. Biol. 4, 10–19 (1997)

    Article  CAS  Google Scholar 

  6. Schmid, F. X. & Blaschek, H. A. Native-like intermediate on the ribonuclease A folding pathway. Eur. J. Biochem. 114, 111–117 (1981)

    Article  CAS  Google Scholar 

  7. Jennings, P. A., Finn, B. E., Jones, B. E. & Matthews, C. R. A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model. Biochemistry 32, 3783–3789 (1993)

    Article  CAS  Google Scholar 

  8. Kamagata, K., Sawano, Y., Tanokura, M. & Kuwajima, K. Multiple parallel-pathway folding of proline-free staphylococcal nuclease. J. Mol. Biol. 332, 1143–1153 (2003)

    Article  CAS  Google Scholar 

  9. Dinner, A. R., Sali, A., Smith, L. J., Dobson, C. M. & Karplus, M. Understanding protein folding via free-energy surfaces from theory and experiment. Trends Biochem. Sci. 25, 331–339 (2000)

    Article  CAS  Google Scholar 

  10. Frieden, C. Slow transitions and hysteretic behavior in enzymes. Annu. Rev. Biochem. 48, 471–489 (1979)

    Article  CAS  Google Scholar 

  11. Flomenbom, O. et al. Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules. Proc. Natl Acad. Sci. USA 102, 2368–2372 (2005)

    Article  ADS  CAS  Google Scholar 

  12. Lu, H. P., Xun, L. & Xie, X. S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998)

    Article  ADS  CAS  Google Scholar 

  13. English, B. P. et al. Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nature Chem. Biol. 2, 87–94 (2005); erratum 2, 168 (2006)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Treiber, D. K. & Williamson, J. R. Exposing the kinetic traps in RNA folding. Curr. Opin. Struct. Biol. 9, 339–345 (1999)

    Article  CAS  Google Scholar 

  16. Chen, S.-J. & Dill, K. A. RNA folding energy landscapes. Proc. Natl Acad. Sci. USA 97, 646–651 (2000)

    Article  ADS  CAS  Google Scholar 

  17. Pan, J., Thirumalai, D. & Woodson, S. A. Folding of RNA involves parallel pathways. J. Mol. Biol. 273, 7–13 (1997)

    Article  CAS  Google Scholar 

  18. Zhuang, X. et al. Correlating structural dynamics and function in single ribozyme molecules. Science 296, 1473–1476 (2002)

    Article  ADS  CAS  Google Scholar 

  19. Tan, E. et al. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate. Proc. Natl Acad. Sci. USA 100, 9308–9313 (2003)

    Article  ADS  CAS  Google Scholar 

  20. Herschlag, D. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry 31, 1386–1399 (1992)

    Article  CAS  Google Scholar 

  21. Bevilacqua, P. C., Kierzek, R., Johnson, K. A. & Turner, D. H. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods. Science 258, 1355–1358 (1992)

    Article  ADS  CAS  Google Scholar 

  22. Ditzler, M. A., Rueda, D., Mo, J., Hakansson, K. & Walter, N. G. A rugged free energy landscape separates multiple functional RNA folds throughout denaturation. Nucleic Acids Res. 36, 7088–7099 (2008)

    Article  CAS  Google Scholar 

  23. Lindahl, T., Adams, A. & Fresco, J. R. Renaturation of transfer ribonucleic acids through site binding of magnesium. Proc. Natl Acad. Sci. USA 55, 941–948 (1966)

    Article  ADS  CAS  Google Scholar 

  24. Korennykh, A. V., Plantinga, M. J., Correll, C. C. & Piccirilli, J. A. Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin. Biochemistry 46, 12744–12756 (2007)

    Article  CAS  Google Scholar 

  25. Levinthal, C. Are there pathways for protein folding? J. Chim. Phys. 65, 44–45 (1968)

    Article  Google Scholar 

  26. Russell, R. et al. Exploring the folding landscape of a structured RNA. Proc. Natl Acad. Sci. USA 99, 155–160 (2002)

    Article  ADS  CAS  Google Scholar 

  27. Zhuang, X. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000)

    Article  ADS  CAS  Google Scholar 

  28. Sattin, B. D., Zhao, W., Travers, K., Chu, S. & Herschlag, D. Direct measurement of tertiary contact cooperativity in RNA folding. J. Am. Chem. Soc. 130, 6085–6087 (2008)

    Article  CAS  Google Scholar 

  29. Russell, R. & Herschlag, D. Probing the folding landscape of the Tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway. J. Mol. Biol. 308, 839–851 (2001)

    Article  CAS  Google Scholar 

  30. Narlikar, G. J., Bartley, L. E., Khosla, M. & Herschlag, D. Characterization of a local folding event of the Tetrahymena group I ribozyme: effects of oligonucleotide substrate length, pH, and temperature on the two substrate binding steps. Biochemistry 38, 14192–14204 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. H. Lee, B. Cui, H. Kim, W. Zhao and other current and former members of the Chu laboratory, and the Mabuchi laboratory, for technical assistance. We thank members of the Herschlag laboratory for discussions and comments on the manuscript. Financial support for this work was provided by US National Institutes of Health (NIH) programme project grant P01-GM-66275 and NIH grant GM49243, to D.H. We thank the Stanford Bio-X Program for fellowship support to S.V.S.

Author Contributions All authors contributed to the experimental design and writing of the manuscript. S.V.S. performed the experiments and M.G. and S.V.S. carried out data analysis.

Author information

Author notes

  1. Steven Chu

    Present address: Present address: United States Department of Energy, Washington DC 20585, USA.,

Authors and Affiliations

  1. Department of Biochemistry,,

    Sergey V. Solomatin & Daniel Herschlag

  2. Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA,

    Max Greenfeld

  3. Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA ,

    Steven Chu

  4. Department of Physics and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA,

    Steven Chu

Authors
  1. Sergey V. Solomatin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Max Greenfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steven Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel Herschlag
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel Herschlag.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1- S7 with Legends, Supplementary Data, Supplementary Table 1 and Supplementary References. (PDF 559 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Solomatin, S., Greenfeld, M., Chu, S. et al. Multiple native states reveal persistent ruggedness of an RNA folding landscape. Nature 463, 681–684 (2010). https://doi.org/10.1038/nature08717

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08717

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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