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While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m⁵U54). Here, we uncover contributions of m⁵U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m⁵U in the T-loop (TrmA inEscherichia coli, Trm2 inSaccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m⁵U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells,trm2Δcell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m⁵U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.