The frameshifting stimulatory element (FSE) is a conserved RNA structure in both SARS-CoV-1 and SARS-CoV-2 that plays a pivotal role in regulating programmed −1 ribosomal frameshifting, a process essential for the synthesis of viral polyproteins required for replication. Structural perturbations to the FSE can markedly alter frameshifting efficiency, thereby disrupting viral propagation and highlighting the FSE as a promising target for antiviral therapeutics. Recent structural predictions indicate that the FSE adopts multiple conformations, each with distinct secondary structures, Although structural plasticity is believed to be crucial for frameshifting, the transition pathways between different FSE RNA conformations remain poorly understood. In this study, we employed Multithermal-Multiumbrella On-the-Fly Probability Enhanced Sampling (MM-OPES) to characterize the conformational landscape of the FSE, focusing on the transition between the 3-stem H-type pseudoknot (motif 3_6) and the HL-type 3-stem pseudoknot (motif 3_3) within an 87-nucleotide (nt) region. Our findings reveal asymmetry in the transition pathways: while multiple routes, both stepwise and cooperative, mediate the transition from motif 3_3 to 3_6, the reverse transition occurs exclusively through a stepwise mechanism. Additionally, we identify the destabilizing role of the flexible 5′ end in motif 3_6, which facilitates its conversion to motif 3_3. These insights highlight the asymmetric nature of FSE conformational transitions and their potential implications for frameshifting regulation.
