Figure 3.

Preparation of synthetic tRNA^Met variants. (A) Process of synthesizing mt tRNA^Met by a combination of chemical synthesis and ligation (33). The synthetic tRNAs have sequences identical to the respective sequences of native tRNA^Met, except for f⁵C and pseudouridine (Ψ). (B) Sequencing analysis using the Donis–Keller′s method of the synthetic mt tRNA^Met labeled with [³²P] at the 5′-end (left) and the 3′-end (right), respectively (30,32). Electrophoresis was performed on a 15% polyacrylamide–7 M urea–10% glycerol gel. Lanes: control without ribonuclease (RNase) (lane 1); limited alkaline hydrolysis (lanes 2 and 7); digestion by RNase T₁ (specific for G: lane 3), RNase U₂ (specific for A: lane 4), RNase PhyM (specific for A and U: lane 5) and RNase CL₃ (specific for C: lane 6). As indicated by the arrows, Ψ was not digested by RNase PhyM (12). The numbering of the residues in the cloverleaf structure of the mt tRNA conforms to that in the previous report (71).