Length-dependent motions of SARS-CoV-2 frameshifting RNA pseudoknot and alternative conformations suggest avenues for frameshifting suppression

The SARS-CoV-2 frameshifting element (FSE), a highly conserved mRNA region required for correct translation of viral polyproteins, defines an excellent therapeutic target against Covid-19. As discovered by our prior graph-theory analysis with SHAPE experiments, the FSE adopts a heterogeneous, length-dependent conformational landscape consisting of an assumed 3-stem H-type pseudoknot (graph motif 3_6), and two alternative motifs (3_3 and 3_5). Here, for the first time, we build and simulate, by microsecond molecular dynamics, 30 models for all three motifs plus motif-stabilizing mutants at different lengths. Our 3_6 pseudoknot systems, which agree with experimental structures, reveal interconvertible L and linear conformations likely related to ribosomal pausing and frameshifting. The 3_6 mutant inhibits this transformation and could hamper frameshifting. Our 3_3 systems exhibit length-dependent stem interactions that point to a potential transition pathway connecting the three motifs during ribosomal elongation. Together, our observations provide new insights into frameshifting mechanisms and anti-viral strategies.