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Frameshifting is an essential mechanism employed by many viruses including coronaviruses to produce viral proteins from a compact RNA genome. It is facilitated by specific RNA folds in the frameshift element (FSE), which has emerged as an important therapeutic target. For SARS-CoV-2, a specific 3-stem pseudoknot has been identified to stimulate frameshifting. However, prior studies and our RNA-As-Graphs analysis coupled to chemical reactivity experiments revealed other folds, including a different pseudoknot. Although structural plasticity has been proposed to play a key role in frameshifting, paths between different FSE RNA folds have not been yet identified. Here, we capture atomic-level transition pathways between two key FSE pseudoknots by transition path sampling coupled to Markov State Modeling and our BOLAS free energy method. We reveal multiple transition paths within a heterogeneous, multihub conformational landscape. A shared folding mechanism involves RNA stem unpairing followed by a 5^′-chain end release. Significantly, this pseudoknot transition critically tunes the tension through the RNA spacer region and places the viral RNA in the narrow ribosomal channel. Our work further explains the role of the alternative pseudoknot in ribosomal pausing and clarifies why the experimentally captured pseudoknot is preferred for frameshifting. Our capturing of this large-scale transition of RNA secondary and tertiary structure highlights the complex pathways of biomolecules and the inherent multifarious aspects that viruses developed to ensure virulence and survival. This enhanced understanding of viral frameshifting also provides insights to target key transitions for therapeutic applications. Our methods are generally applicable to other large-scale biomolecular transitions. 

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed −1 ribosomal frameshift (−1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.