tRNA binding and modification by the Escherichia coli dihydrouridine synthase DusA

Dihydrouridine (D) is one of the most highly conserved tRNA modifications throughout all domains of life. D20 is particularly conserved and is formed by DusA in E. coli, but its mechanisms and functions remain uncovered. Here, we characterized the molecular mechanisms of DusA in tRNA binding, cofactor oxidation, and tRNA modification, in addition to the cellular functions of DusA in tRNA maturation and mRNA translation. Thereby, we reveal DusA binds tRNA using a two-step mechanism involving local tRNA conformational changes. Although DusA can modify an unmodified tRNA substrate, it binds to previously modified tRNA with a higher affinity. In contrast to the well-studied T arm modifying enzymes TrmA and TruB, DusA does not globally increase tRNA aminoacylation levels, but does improve aminoacylation for specific tRNA species. Despite only few changes in tRNA charging, DusA improves the translation for several specific codons, suggesting a direct role for dihydrouridine in tRNA function on the ribosome. Thus, we propose DusA functions non-redundantly with TrmA and TruB to fine-tune protein synthesis. Overall design: Total RNA samples were extracted from early log phase WT (n=3) and ?dusA (n=3) E. coli strain BW25113 under acidic conditions (pH <5) using TRIzol. Samples were sent to MesoRNA on dry ice where they were underwent periodate oxidation/beta elimination, library preparation, and sequencing as described previously (PMID: 35513407).