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Evolution of Tetrahymena ribozyme mutants with increased structural stability

Nature Structural Biology volume 9, pages 855–861 (2002)Cite this article

Abstract

Determining how large RNA molecules stabilize their tertiary structures is critical for understanding how they perform their biological functions. Here we use in vitro selection to identify active variants of the Tetrahymena ribozyme with increased stability. The mutant pool converged to a single family that shared nine mutations; an RNArepresenting the consensus sequence was structurally more stable by 10.5 °C and catalytically active at elevated temperatures. Remarkably, of the nine altered sites, most are already known to be involved in tertiary interactions, and the stabilizing mutations primarily improve the packing interactions in the molecular interior. The wild type ribozyme and the selected mutants provide pairs of mesophilic and thermophilic homologs for studying the origin of their thermal stability.

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Figure 1: In vitro selection for stability and activity.
Figure 2: Selection progression.
Figure 3: Location of the consensus mutations.
Figure 4: TGGE comparing R14C with the wild type ribozyme (WT).
Figure 5: UV melting of the wild type ribozyme and the consensus mutants.
Figure 6: Selected mutants retain enzymatic activity at high temperature.

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Acknowledgements

We are grateful to H. Brummel and K. Kossen for help with the UV melting experiments. We thank A. Gooding for providing T7 RNA polymerase and E. Podell and K. Goodrich for oligonucleotide synthesis. We also thank A. Zaug, A. Berglund, E. Podell, P. Baumann and G. Joyce for insightful discussions.

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  1. Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, 80309-0215, Colorado, USA

    Feng Guo & Thomas R. Cech

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  1. Feng Guo
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Correspondence to Thomas R. Cech.

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Guo, F., Cech, T. Evolution of Tetrahymena ribozyme mutants with increased structural stability. Nat Struct Mol Biol 9, 855–861 (2002). https://doi.org/10.1038/nsb850

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