All elongator tRNAs harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, which are generated by the enzymes TrmA and TruB, respectively.Escherichia coliTrmA and TruB both act as tRNA chaperones, and strains lackingtrmAortruBare outcompeted by wildtype. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion oftrmAandtruBinE. colicauses a global decrease in aminoacylation and alters other tRNA modification such as acp3U47. Whereas overall protein synthesis is not affected in ΔtrmAand ΔtruBstrains, the translation of a specific subset of codons is significantly impaired, and the expression of many specific proteins is translationally changed. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness and explainingtrmAandtruBconservation.