Site-Specific Profiling of 4-Thiouridine Across Transfer RNA Genes in Escherichia coli

[Image: see text] The transfer RNA (tRNA) modification 4-thiouridine (s(4)U) acts as a near-ultraviolet (UVA) radiation sensor in Escherichia coli (E. coli), where it induces a growth delay upon exposure to the UVA radiation (∼310–400 nm). Herein, we report sequencing methodology for site-specific profiling of s(4)U modification in E. coli tRNAs. Upon the addition of iodoacetamide (IA) or iodoacetyl-PEG2-biotin (BIA), the nucleophilic sulfur of s(4)U forms a reaction product that is extensively characterized by liquid chromatography–mass spectrometry (LC–MS/MS) analysis. This method is readily applied to the alkylation of natively occurring s(4)U on E. coli tRNA. Next-generation sequencing of BIA-treated tRNA from E. coli revealed misincorporations at position 8 in 19 of the 20 amino acid tRNA species. Alternatively, tRNA from the ΔthiI strain, which cannot introduce the s(4)U modification, does not exhibit any misincorporation at the corresponding positions, directly linking the base transitions and the tRNA modification. Independently, the s(4)U modification on E. coli tRNA was further validated by LC–MS/MS sequencing. Nuclease digestion of wild-type and deletion strains E. coli tRNA with RNase T1 generated smaller s(4)U/U containing fragments that could be analyzed by MS/MS analysis for modification assignment. Furthermore, RNase T1 digestion of tRNAs treated either with IA or BIA showed the specificity of iodoacetamide reagents toward s(4)U in the context of complex tRNA modifications. Overall, these results demonstrate the utility of the alkylation of s(4)U in the site-specific profiling of the modified base in native cellular tRNA.