Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 μs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 μs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.
RNA pseudoknots compose a three‐dimensional structural motif that is present in the catalytic cores of some ribozymes, and are also capable of stimulating ribosomal frameshifts. Furthermore, their complex topology and non‐canonical hairpin‐loop composition make pseudoknots an essential structural motif with which to study the RNA folding process. Here we report our analysis of nearly 20,000 independent all‐atom molecular dynamics simulations of the ribosomal frame‐shifting pseudoknot of Luteovirus and the tmRNA pseudoknot from Aquifex aeolicus, which share global topology but have only ~50% sequence similarity. Using the Folding@Home distributed computing network and our new Pathway Enumeration sampling method, a cumulative sampling time of over 100 μs was achieved for each of these pseudoknots. K‐means clustering was used to identify 27 conformational microstates for each pseudoknot and similar folding pathways were identified for these two sequences. In agreement with our earlier work, this study suggests that native state topology is a predominate factor in the RNA folding mechanism.This work was made possible by the worldwide Folding@Home volunteers who contributed invaluable processor time, Women & Philanthropy, and a Research Corporation Cottrell College Science Award.
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