Mapping the temporality and structural impacts of modifications in Escherichia coli tRNAs

Transfer RNAs (tRNAs) are essential components of the protein synthesis machinery. Their biogenesis is a highly regulated process that involves the incorporation of numerous post-transcriptional chemical modifications, essential for tRNA folding, cellular stability and function. The sequential process by which these modifications are introduced remains poorly characterized. Previous studies have suggested the existence of modification hierarchies, particularly in the anticodon-loop region, but also among tRNA core modifications. Here, aiming to understand the molecular mechanisms by which modifications are incorporated in a bacterial model organism, we employed a combination of NMR spectroscopy and biochemical methods to characterize the maturation process of several Escherichia coli tRNAs. By monitoring tRNA maturation in a time-resolved fashion by NMR, we observed a conserved temporal pattern in the incorporation of the Ψ55, T54, and m⁷G46 modifications. We also show that Ψ55 stimulates the incorporation of T54 in E. coli tRNA^Phe, tRNA^Val, and tRNA^Asp and stimulates that of m⁷G46 in tRNA^Phe and tRNA^Asp. Importantly, we also provide general insights into the impact of modifications on tRNA structural properties and show that while post-transcriptional modifications generally have a structuring effect that reduces conformational heterogeneities, these effects are tRNA-dependent, with certain tRNAs being more affected than others. These findings provide fundamental insights into the molecular aspects of tRNA maturation in E. coli.