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; 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.
Highlights
The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target
We examine the conformational dynamics of the SARSCoV-2 pseudoknot in the single-molecule regime. We study it under tension in optical tweezers in order to mimic the situation seen during −1 PRF, where the force applied by the ribosome is ramped up and down as the ribosome attempts to resolve the mRNA structure before shifting reading frame[30]
To probe the conformations formed by the SARS-CoV-2 pseudoknot, their folding pathways, and the dynamics under tension, we annealed a single RNA molecule containing the sequence of the pseudoknot flanked by handle regions to DNA handles that were attached to beads held in optical traps (Fig. 2a)
Summary
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. CryoEM imaging[10,15] and computational modeling[18] both suggest that the SARS-CoV-2 pseudoknot can take on several different conformers (Fig. 1b, c) Some of these conformers involve knot-like fold topologies that have not previously been observed in frameshiftstimulatory pseudoknots, conformers with the 5′ end threaded through the junction between the three helices to generate what we term a ring-knot[10,15,18]. The dynamic ensemble of conformers populated by the SARS-CoV-2 pseudoknot has not yet been explored experimentally, and the folding mechanism of this pseudoknot—especially its unusual ring-knotted conformer— remains unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
