RNA Structural Ensemble Determinants of −1 Programmed Ribosomal Frameshifting Efficiency Across Coronavirus Evolution

Coronavirus -1 programmed ribosomal frameshifting (-1 PRF) elements adopt complex structural ensembles that are essential for maintaining efficient frameshifting. Here we study four novel -1 PRF elements to experimentally determine and characterize their structural ensembles. Coupling this structural information with measurements of -1 PRF efficiency provides insights into the structural features responsible for refining rates of -1 PRF in better versus worse frameshifting elements (FSEs). The structure/function relationships that were previously found to govern -1 PRF efficiency in SARS-CoV-2 do not fully generalize in other FSEs. Specifically, the observation that -1 PRF efficiency increases with the number of alternative conformations is not always the case. One of the most efficient -1 PRF elements in this study, the BtRs-BetaCoV element, adopts a single conformation. We find that no single structural feature, including the relative strength of the attenuator hairpin, is predictive of frameshifting efficiency. However, we do observe a trend away from canonical stem I formation in better frameshifting elements despite reports of strong selective pressures. Finally, by swapping viral sequence elements in a modular manner, we reveal how sequence elements upstream of the attenuator hairpin substantially enhance or suppress viral frameshifting. This study provides detailed characterization of short frameshifting elements that may have biotechnological applications in packaging gene therapies or controlling stoichiometric protein expression ratios.