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; Mg2+ 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.
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; n class="Chemical">Mg2+ 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.
Authors: John F Atkins; Gary Loughran; Pramod R Bhatt; Andrew E Firth; Pavel V Baranov Journal: Nucleic Acids Res Date: 2016-07-19 Impact factor: 16.971
Authors: Thomas A Hilimire; Jeffrey M Chamberlain; Viktoriya Anokhina; Ryan P Bennett; Oliver Swart; Jason R Myers; John M Ashton; Ryan A Stewart; Aaron L Featherston; Kathleen Gates; Eric D Helms; Harold C Smith; Stephen Dewhurst; Benjamin L Miller Journal: ACS Chem Biol Date: 2017-05-05 Impact factor: 5.100
Authors: Jamie A Kelly; Alexandra N Olson; Krishna Neupane; Sneha Munshi; Josue San Emeterio; Lois Pollack; Michael T Woodside; Jonathan D Dinman Journal: J Biol Chem Date: 2020-06-22 Impact factor: 5.157
Authors: Sneha Munshi; Krishna Neupane; Sandaru M Ileperuma; Matthew T J Halma; Jamie A Kelly; Clarissa F Halpern; Jonathan D Dinman; Sarah Loerch; Michael T Woodside Journal: Viruses Date: 2022-01-18 Impact factor: 5.818
Authors: Tammy C T Lan; Matty F Allan; Lauren E Malsick; Jia Z Woo; Chi Zhu; Fengrui Zhang; Stuti Khandwala; Sherry S Y Nyeo; Yu Sun; Junjie U Guo; Mark Bathe; Anders Näär; Anthony Griffiths; Silvi Rouskin Journal: Nat Commun Date: 2022-03-02 Impact factor: 14.919