Thermodynamics and Kinetics of the Hairpin Ribozyme from Atomistic Folding/Unfolding Simulations

Lucas G Nivón,Eugene I Shakhnovich

doi:10.1016/j.jmb.2011.06.042

Abstract

We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here, we introduce a simple model of tertiary-structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary-structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition-state ensemble (TSE) of the RNA during folding to its active “docked” conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters, we introduce a novel method to benchmark Monte Carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential that discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures.

Highlights

Ribozymes perform a variety of catalytic functions in nature and the exact three-dimensional structure in the vicinity of the active site is responsible for the vast rate-enhancements affected by RNA
We expect structures encountered in time immediately prior to folding to be members of the transition state ensemble (TSE)
We can ask the question, for each structure, is this truly a member of the TSE? If a structure is in the TSE, it should have 50% probability of docking rapidly and 50% probably of undocking – post-TSE structures have close to 100% probability of folding, while pre-TSE structures have close to 0% probability

Summary

Introduction

Ribozymes perform a variety of catalytic functions in nature and the exact three-dimensional structure in the vicinity of the active site is responsible for the vast rate-enhancements affected by RNA. It is of great interest to understand the exact sequence of events through which a newly synthesized RNA chain folds into the catalytically active native state before commencing cleavage, ligation, or one of many other catalytic roles. Efforts to understand RNA folding mechanism have focused largely on auto-catalytic introns, such as the Group I intron from Tetrahymena, and on smaller self-cleaving RNAs such as the hairpin, hammerhead, hepatitis delta, and Neurospora VS ribozymes.[1,2] The hairpin ribozyme was discovered in the negative strand of tobacco ringspot virus roughly twenty years ago.[3,4] It has at least two domains of secondary structure joined by tertiary interactions in the active form.[5,6] In the minimal, two-domain form, this ribozyme provides an ideal model for the formation of tertiary structure in RNA folding because it has so few

Results

Discussion

Conclusion