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bioRxiv · 10.1101/2025.05.06.652307

A pseudouridine synthase shapes tRNA structural dynamics through both catalysis and remodeling

Dennis, E. M.·Nieves, N. E. C.·Vaaler, A. L.·Kadrmas, M.·Barry, M. L.·Garcia, D. M.·Widom, J. R.
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Abstract

Transfer RNA has long served as an exemplar of a thermodynamically stable, structured RNA. Yet it undergoes significant structural changes upon binding and catalysis by diverse modification enzymes. We leveraged optical binding assays and single-molecule FRET to observe the structural dynamics of two yeast tRNAs, in isolation, and upon interaction with the conserved pseudouridine synthase Pus4/TruB. We show that unmodified and pseudouridylated tRNAeMet(CAU) and tRNAThr(AGU) all sample open, compact and intermediate conformations, though tRNAThr(AGU) exhibits faster dynamics. Consistent with its role in modifying RNAs with different structural properties, Pus4 binds robustly to unmodified tRNA, tRNA that was pseudouridylated prior to engaging Pus4 ("pre-modified"), and even an unrelated riboswitch RNA. Pus4 binding to tRNAeMet(CAU) leads to additional tRNA conformational states. The ensemble of conformations explored by Pus4-bound pre-modified tRNA resolved within minutes back into open, compact and intermediate states. tRNAeMet(CAU) that is initially unmodified more gradually approached the ensemble of structures that were attained rapidly by pre-modified tRNA. Thus, Pus4 both catalyzes a lasting chemical change on tRNA and remodels it over time after catalysis, perhaps to promote subsequent steps of tRNA maturation.

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Dennis, E. M., Nieves, N. E. C., Vaaler, A. L., Kadrmas, M., Barry, M. L., Garcia, D. M., Widom, J. R.. 2025-05-09. A pseudouridine synthase shapes tRNA structural dynamics through both catalysis and remodeling. https://doi.org/10.1101/2025.05.06.652307

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