The tRNA dihydrouridine synthase DusA has a distinct mechanism in optimizing tRNAs for translation

Dihydrouridine (D) is one of the most highly conserved RNA modifications across all domains of life. D20 within the tRNA D loop is particularly conserved and is formed by DusA in Escherichia coli. However, the mechanisms and cellular functions of DusA and D20 remain poorly understood. Here, we characterize DusA's role in tRNA binding, cofactor oxidation, and modification activity, along with its impact on tRNA maturation and translation. We find that DusA binds tRNA via a two-step mechanism involving a local structural rearrangement and exhibits a higher affinity for previously modified tRNA compared to unmodified tRNA. Unlike the T arm modifying enzymes TrmA and TruB, DusA does not broadly increase cellular aminoacylation for all tRNAs but enhances the charging of specific tRNA species. Despite limited alterations in overall tRNA charging and abundance in cells lacking DusA, DusA selectively improves translation at several specific codons, potentially indicating a direct contribution for dihydrouridine to the function of certain tRNAs on the ribosome. In conclusion, our findings suggest DusA acts nonredundantly with and complementary to TrmA and TruB in fine-tuning protein synthesis.