Mechanical Force and Salt Effects on the Thermodynamics of a Frameshifting RNA Pseudoknot

Abstract

Mechanical resistance of pseudoknot (PK) in mRNA deemed relevant in stimulating programmed ribosomal frameshifting (PRF) has lead to a number of single molecule pulling experiments in order to decipher the PRF mechanism. Motivated by these studies, we performed simulations to describe the response of a Beet Western Yellow Virus (BWYV) PK over a range of mechanical forces (fs) and monovalent salt concentrations (Cs) using a coarse-grained model. The simulations based on the model quantitatively reproduces the three-stage thermal melting of BWYV PK and the stability of the folded state (F) with respect to the unfolded state (U) inferred in ensemble experiments setting the stage for making testable predictions. The predicted phase diagram in the [C, f] plane shows that a sequence of structural transitions populating intermediates occur as f and C are changed. The least stable tertiary interactions rupture first followed by unfolding of the 3′-end hairpin. Finally, the most stable 5′-end hairpin unravels producing a stretched state. A theoretical analysis of the phase boundaries, separating the F and an intermediate state (I), and I and U states shows that the critical force for rupture scales as (log Cm)α with α=1 (=0.5) for U−I (I−F) transition. This relation is used to obtain the ion-preferential coefficients, which suggests that ion-RNA interactions can be quantitatively measured using laser optical tweezers experiments. We predict that −1 PRF efficiency should be determined by the stability of the 5′ hairpin that the ribosome first encounters. A good correlation between the increase in −1 PRF efficiency and increase in the stability of 5′ hairpin supports this prediction.