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

TL;DR

This study shows how a tRNA-modifying enzyme, Pus4, alters tRNA structure both by adding a chemical modification and by reshaping its conformation over time.

Contribution

The paper reveals that Pus4 influences tRNA structure through both catalytic modification and dynamic remodeling.

Findings

Unmodified and modified tRNA sample similar dynamic conformations.

Pus4 binding introduces new conformational states in unmodified tRNA.

A catalytically inactive Pus4 mutant alters tRNA conformations differently than the active enzyme.

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 tRNA structural dynamics before and upon interaction with the conserved pseudouridine synthase Pus4/TruB. We show that unmodified and pseudouridylated tRNA similarly sample one open and two closed conformations, dynamically. Binding by Pus4 to unmodified tRNA populates additional conformational states, gradually approaching an ensemble that is adopted sooner by tRNA that was pseudouridylated prior to engaging Pus4. A catalytically incompetent mutant of Pus4 binds more slowly and remodels unmodified and pre-modified tRNAs into different conformational ensembles than wild-type enzyme. Thus, Pus4 both catalyzes a lasting chemical change on tRNA and remodels it over time, perhaps to advance subsequent steps of tRNA maturation.