Introduction

Materials and Methods

Results and Discussion

Conclusions

References

ABSTRACT

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg2þ, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg2þ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg2þ-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg2þ, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg2þ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg2þ-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg2þ-ion interactions contribute to RNA structural properties and stability.

INTRODUCTION

RNA molecules have highly diverse structures ranging from simple helices to highly heterogeneous folded conformations that are essential for their wide range of cellular functions (1–۳). Specifically, ribozymes, a distinct class of enzymes, exhibit complex tertiary structures and catalyze self-cleavage or the cleavage of phosphodiester bonds of substrate RNA, with metal ions typically playing a central role in the catalytic activity (4–۶). To assume their tertiary structures, RNAs must overcome large unfavorable electrostatic interactions associated with their polyanionic phosphodiester backbone (7). To facilitate this, positively charged ions screen the highly negative potential, allowing the RNA secondary structures to collapse into compact tertiary conformations (8–۱۱). Typically divalent ions, most often Mg2þ, facilitate the formation of tertiary interactions required for the full folding of RNA (12–۱۴). However, the inability to visualize the ions during folding represents a key barrier to understanding the role of divalent ions in folding of RNA (15). Studies have used classical molecular dynamics (MD) or other theoretical approaches to investigate Mg2þ-RNA binding, but they were limited to native conformations because of their inability to overcome the issues associated with the Mg2þ exchange rates (16–۲۲). The exchange rate of water complexed to Mg2þ is on the ms timescale (6.7 105 s 1 ), and the exchange rate of Mg2þ with phosphate is on the ms timescale (0.5–۲٫۵ ۱۰۳ s 1 ) (23), which is beyond the timescale of typical atomistic MD simulations (20,24) such that only limited insights into Mg2þ-RNA interactions are accessible (25). Alternatively, simulations using coarse-grained models of nucleic acids provided insights into how Mg2þ can serve to nucleate the folding of key tertiary interactions with the Mg2þ-RNA interactions being dominated by specific interactions even in unfolded states (19,26).