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

The transfer RNA
(tRNA) modification 4-thiouridine (s4U) 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 s4U modification in E. coli tRNAs. Upon the addition of iodoacetamide
(IA) or iodoacetyl-PEG2-biotin (BIA), the nucleophilic sulfur of s4U 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 s4U 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
s4U modification, does not exhibit any misincorporation
at the corresponding positions, directly linking the base transitions
and the tRNA modification. Independently, the s4U 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 s4U/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 s4U in the
context of complex tRNA modifications. Overall, these results demonstrate
the utility of the alkylation of s4U in the site-specific
profiling of the modified base in native cellular tRNA.