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Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation

Nature Structural Biology volume 7, pages 371–374 (2000)Cite this article

Abstract

For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods. We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme. The cooperativity of folding, m-value, and the free energy, ΔG°N−U, associated with formation of each tertiary contact was determined by analysis of the isotherms. The ΔG°N−U values measured in this study vary from 1.7 ± 0.2 to 7.6 ± 1.2 kcal mol−1. Thus, the stability of these discrete tertiary contacts vary by almost 104. In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 ± 0.2 to 1.7 ± 0.3 kcal mol−1 M−1) indicating that these contacts exhibit global cooperatively in their folding behavior. This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.

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Figure 1: Urea-induced unfolding of the Tetrahymena ribozyme.
Figure 2: Free energy and m-values derived for individual sites in the Tetrahymena ribozyme.
Figure 3: Individual site free energy and m-values derived for the isolated P4-P6 domain.

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Acknowledgements

We are grateful to S. Woodson for providing the DNA templates used for transcription of the Tetrahymena ribozyme and the P4-P6 subdomain of the ribozyme. We also thank M. Deras and J. Swisher for advice and help in the preparation and handling of RNA samples. This work was supported by grants from the NIH Institute for General Medical Sciences and the Biomedical Technology Program of the Division of Research Resources. The National Synchrotron Light Source at Brookhaven National Laboratory is supported by the Department of Energy.

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Authors and Affiliations

  1. Center for Synchrotron Biosciences, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Corie Y. Ralston, Qin He, Michael Brenowitz & Mark R. Chance

  2. Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Corie Y. Ralston, Qin He & Mark R. Chance

  3. Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Michael Brenowitz & Mark R. Chance

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  1. Corie Y. Ralston
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Correspondence to Mark R. Chance.

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Ralston, C., He, Q., Brenowitz, M. et al. Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation. Nat Struct Mol Biol 7, 371–374 (2000). https://doi.org/10.1038/75139

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