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

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.