Figure 2.
Methylation activity assays. The unmodified human mitochondrial wildtype (A37) and mutant (G37) tRNAMet, cytosolic tRNALeu(CAG), and tRNAThr were generated from in vitro transcription. A, analysis for the m1G37 modification of tRNAMet. 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 Samples were withdrawn and stopped after 2, 4, 6, or 8 min, respectively.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. Graph shows the results of a representative experiment. B, electrophoretic mobility shift assay. The unmodified mitochondrial wildtype (A37) and mutant (G37) tRNAMet were incubated with various concentrations of enzymes Mj-Trm5. These samples were electrophoresed through 6% polyacrylamide gel and stained with ethidium bromide.