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Thermodynamics of unfolding mechanisms of mouse mammary tumor virus pseudoknot from a coarse-grained loop-entropy model

Abstract

Pseudoknotted RNA molecules play important biological roles that depend on their folded structure. To understand the underlying principles that determine their thermodynamics and folding/unfolding mechanisms, we carried out a study on a variant of the mouse mammary tumor virus pseudoknotted RNA (VPK), a widely studied model system for RNA pseudoknots. Our method is based on a coarse-grained discrete-state model and the algorithm of PK3D (pseudoknot structure predictor in three-dimensional space), with RNA loops explicitly constructed and their conformational entropic effects incorporated. Our loop entropy calculations are validated by accurately capturing previously measured melting temperatures of RNA hairpins with varying loop lengths. For each of the hairpins that constitutes the VPK, we identified alternative conformations that are more stable than the hairpin structures at low temperatures and predicted their populations at different temperatures. Our predictions were validated by thermodynamic experiments on these hairpins. We further computed the heat capacity profiles of VPK, which are in excellent agreement with available experimental data. Notably, our model provides detailed information on the unfolding mechanisms of pseudoknotted RNA. Analysis of the distribution of base-pairing probability of VPK reveals a cooperative unfolding mechanism instead of a simple sequential unfolding of first one stem and then the other. Specifically, we find a simultaneous “loosening” of both stems as the temperature is raised, whereby both stems become partially melted and co-exist during the unfolding process.

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Acknowledgements

We thank Drs. Youfang Cao, David Jimenez Morales, Hammad Naveed, Hsiao-Mei Lu, Yun Xu, Gamze Gursoy, Meishan Lin, Anna Terebus, Wei Tian and Jieling Zhao for helpful discussions. We are deeply indebted to Hans Frauenfelder and his colleagues for introducing the framework of energy landscapes and conformational substates of biological molecules. These concepts have had a profound impact on how we approach the problems of structure-dynamics-function relationships in biology.

Funding

This work was supported by grants from the National Institutes of Health (R35 GM127084) to J.L. and National Science Foundation (MCB-1158217 and MCB-1715649) to A.A.

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Tang, K., Roca, J., Chen, R. et al. Thermodynamics of unfolding mechanisms of mouse mammary tumor virus pseudoknot from a coarse-grained loop-entropy model. J Biol Phys 48, 129–150 (2022). https://doi.org/10.1007/s10867-022-09602-2

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  • DOI: https://doi.org/10.1007/s10867-022-09602-2

Keywords

  • RNA folding
  • Pseudoknotted RNA
  • Loop entropy
  • PK3D
  • Unfolding mechanism
  • Energy landscape
  • RNA hairpin
  • Heat capacity
  • Fluorescence spectroscopy