Chronology of tRNA structural dynamics prior to and during interaction with a pseudouridine synthase

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 tRNA^eMet(CAU) and tRNA^Thr(AGU) all sample open, compact, and intermediate conformations, though tRNA^Thr(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 ("premodified"), and even an unrelated riboswitch RNA. Pus4 binding to tRNA^eMet(CAU) leads to additional tRNA conformational states. The ensemble of conformations explored by Pus4-bound premodified tRNA resolved within minutes back into open, compact, and intermediate states. tRNA^eMet(CAU) that is initially unmodified more gradually approached the ensemble of structures that were attained rapidly by premodified 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.