Heterogeneous and multiple conformational transition pathways between pseudoknots of the SARS-CoV-2 frameshift element

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.