Figure 3.

tRNA-MaP successfully detects m¹A sites in tRNA transcripts methylated by recombinant TrmK in vitro.A, workflow of in vitro T7 transcription to prepare a mixture of 59 species of Geobacillus strearothermophilus tRNA transcripts. Aquifex aeolicus tRNAs were also prepared separately. The detailed protocol is provided in the Experimental procedures section. In brief, the tRNA-MaP experiment was performed in triplicates (n = 3). The TrmK reaction was conducted in the presence of 100 nM TrmK, 2 μM tRNA mixture, and 10 μM AdoMet at 60 °C for 10 min, whereas the TrmD reaction was performed in the presence of 400 nM TrmD, 2 μM tRNA mixture, and 10 μM AdoMet at 55 °C for 5 min. B, mutation rates from G. stearothermophilus tRNAs (methylated with TrmK) and A. aeolicus tRNAs (methylated with TrmD) upon analysis by tRNA-MaP are provided. C, the mutation rates at A22 in 23 tRNAs are provided. Errors are SD from three biological replicates (n = 3). MaP, Mutational Profiling.