Simulated and Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

Abstract

Mechanical unfolding of −1 frameshift signals such as RNA pseudoknots have aimed to test the hypothesis that the stability of the pseudoknot (PK) is directly correlated to the frameshifting efficiency. Here we report unfolding of the Beet Western Yellow Virus (BWYV) PK by optical tweezers complemented by computer simulations using steered molecular dynamics (SMD). Three BWYV PK scenarios were studied: the wild-type PK in the presence and absence of Mg2+, and mutations of nucleic base C8 known to completely abolish −1 frameshifting by disrupting pseudoknot stability at the core of its structure. Despite significant differences in loading rates, we found the experimental and computational results to be remarkably consistent.The SMD simulations provide a detailed sequence of molecular unfolding events that can be assigned to the force-extension profiles obtained with the optical tweezers. In the absence of Mg2+, stretching of the PK using the optical tweezers does not result in the observation of any unfolding transitions, which is consistent with the SMD simulation that demonstrates the essential role of Mg2+ for the formation of a very strong salt bridge between G4, C5, G16, and C17 nucleotides. The C8 mutants, like wild-type unfolding in the absence of Mg2+, unfold readily and at low force, consistent with the absence of any −1 frameshifting activity for these mutants.