RedOx  E.coli tRNA D
Various tRNA modifications have recently been shown to regulate gene expression through modulation of mRNA translation, often in the context of stress stimuli. Among these modifications, dihydrouridine stands out for its increase of tRNA structural flexibility. However, if and how dihydrouridine synthesis reacts to environmental stimuli is largely unknown. In this study, we manipulated the intracellular redox state of E. coli using paraquat, revealing differential sensitivities of the three tRNA-dihydrouridine synthases towards oxidative stress. Using LC-MS quantification of dihydrouridine in various knockout strains, we validated the use of a specific RNAseq method, namely AlkAnilineSeq, for the precise mapping of dihydrouridines throughout E. coli tRNAs. We found DusA showing high activity, followed by DusB and DusC, whose activity was decreased under paraquat treatment. The relative sensitivity is most plausibly explained by a paraquat-dependent drop of NADPH availability. These findings are substantiated by in vitro kinetics, revealing that DusA is the most active enzyme, followed by DusB, while DusC showed little activity, likely related to the efficacy of the redox reaction of the flavin coenzyme with NADPH. Overall, our study underscores the intricate interplay between redox dynamics and tRNA modification processes, revealing a new facet of the regulatory mechanisms influencing cellular responses to oxidative stress.