Fig. 5.
Levels of tRNA modification vary under heat stress conditions. (A–D) Normalized reactivity for E. coli tRNA modifications was detected by mutational profiling. “Experiment” shows positions that have high mutation rate (–DMS) whereas “Model” shows positions of the known modification in E. coli. Examples of tRNA mutational profiling for (A) tRNAGly CCC-1–1, (B) tRNALeu TAA-1–1, (C) tRNAThr GGT-1–1, and (D) tRNATyr GTA-1–1 are provided. (E) Mutational profiling is depicted on the secondary structure of tRNA. In the gray box, “known mods” (the denominator) is the number of known natural modifications at particular positions in tRNA according to the Modomics database. The “detected mods” (the numerator) is the number of detected natural modifications by tRNA structure-seq, where detected normalized mutation rates are higher than 2 at the positions. (F) Mutation rates and MS normalized peak areas are correlated. tRNA modification levels estimated by averaged mutational profiling (n = 2) and averaged nucleoside analysis (n = 4). (G–N) Normalized reactivities for overall levels of the known modifications in 36 E. coli tRNAs are plotted. The blue boxes are the standard conditions and the red boxes are the heat conditions. Numerous changes in the individual levels of (G) s4U8, (H) dihydrouridines, (I) Gm18, (J) anticodon modifications at position 32 including Cm32, Um32, and s2C, (K) anticodon modifications at position 34 including cmo5U, Q, ac4C, GluQ, cmnm5Um, mnm5U, cmnm5s2U, mnm5s2U, and Cm, (L) anticodon modifications at position 37 including m2A, ms2i6A, t6A, m6t6A, and m1G, (M) m7G46, and (N) acp3U47 are apparent. All data are found in SI Appendix, Fig. S12.