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. 2021 Jan 4;49(2):1075–1093. doi: 10.1093/nar/gkaa1225

Figure 2.

Figure 2.

The m.4295A>G mutation introduced the m1G37 modification of tRNAIle. (A) Schematic of methylation shown in the cloverleaf structures of the human mitochondrial tRNAIle. An arrow denotes the location of the m.4295A>G mutation. Solid lines represent the DIG-labeled oligonucleotide probe specific for mt-tRNAIle. Broken lines represent the potential stops of primer extension and m1G or m2G. (B) Primer extension demonstrated the creation of m1G37 in the tRNAIle carrying the m.4295A>G mutation. The primer extension termination products are showed as m1G9, m22G26 and m1G37. (C) Methylation activity assays. The unmodified human mitochondrial wild type (A37) and mutant (G37) tRNAIle, cytosolic tRNALeu(CAG) and tRNAThr were generated from in vitro transcription. The unmodified tRNA transcripts were incubated with M. jannaschii (Mj-Trm5) in the presence of S-adenosyl-l-methionine. Samples were withdrawn and stopped after 2, 4, 6 or 8 min, respectively. The relative modification efficiency was calculated from the initial phase of the reaction. The calculations were based on three independent determinations.