Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers
Krishna Neupane, Meng Zhao, Aaron Lyons, Sneha Munshi, Sandaru M. Ileperuma, Dustin B. Ritchie, Noel Q. Hoffer, Abhishek Narayan, Michael T. Woodside
Abstract
Abstract The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5′ end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5′ end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg 2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.