tRNA^Val allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei

Mass spectrometry analysis of the native L. casei tRNA^Val. (A) The MALDI-TOF mass spectrum of the native L. casei tRNA^Val digested by RNase T1 shows s⁴U8 containing fragment AUs⁴UAGp, D containing fragment (UD)Gp, m⁶A37 containing fragment CCUUACm⁶AAGp, m⁷G46 containing fragment m⁷GUCACAGp, and m⁵U54 containing fragment m⁵UUCGp. (B) The MALDI-TOF mass spectrum of the native L. casei tRNA^Val digested by RNase A shows s⁴U8, m⁶A37, m⁷G46, and m⁵U54. Supplemental Tables S1 and S2 give theoretical and empirical masses of singly protonated ions derived from the RNase T1 and RNase A fragments of the L. casei tRNA^Val, respectively. (C) LC-HRMS analysis of nuclease P1 digested L. casei tRNA^Val nucleosides. Overlay of chromatograms obtained for A, C, G, U, Ψ, (D), m⁶A, and s⁴U detected at m/z of 268.1040, 244.0928, 284.0989, 245.0768 (U and Ψ), 247.0925, 282.1197, and 261.0540, respectively, at the corresponding retention time expected from the authentic nucleoside samples. Ions at m/z of 259 and 298 are annotated as m⁵U and m⁷G, respectively. LC-HRMS data for s⁴U is provided in the inset. (D) Modified nucleosides of the native L. casei tRNA^Val detected by mass spectrometry analyses. Detected modified nucleosides and their positions are shown in red letters.