Fig. 1: Leader method for 5’-end processing.

A General scheme of tRNA synthesis coupled with translation in the tfPURE system. The reaction mixture contains tRNA-encoding DNA template (tRNA template) and luciferase-encoding DNA template (luciferase template). The luciferase is translated depending on the synthesized tRNA. B Scheme of 5’-end cleavage by RNase P, using the leader method. A premature tRNA is synthesized with a leader sequence at the 5’end. The leader sequence is removed by RNase P to produce a mature tRNA. M1 RNA, the catalytic RNA component of RNase P, was used for this experiment. C Specific scheme for non-G-start tRNA processing in the tfPURE system. One of the six non-G-start tRNAs was expressed from a tRNA template and cleaved with RNase P. The resultant mature tRNA is used for the translation of luciferase with the other 20 IVS tRNAs. The 3’ end of tRNA was prepared by run-off transcription. D Luciferase activity in the translation-coupled reaction of expressed tRNA. The reaction mixture contained each of six tRNA templates (50 nM), luciferase template (1 nM), RNase P (M1 RNA, 500 nM), T7 RNA polymerase (1.7 μ/μL), 20 IVS tRNAs (excluding the tRNA to be expressed), and the tfPURE system (composition A, Supplementary Table 2). After incubation at 30 °C for 16 h, luminescence was measured. Data are presented as mean values, each data point represents three independent experiments, and error bars indicate standard deviations. E PAGE analysis after the translation-coupled reaction of tRNA^Gln. The reaction was conducted as shown in (D), except for the omission of 20 IVS tRNAs. The bands for uncleaved pre-tRNAs are indicated by blue arrowheads. RNA was stained with SYBR Green II. F PAGE analysis of the other five tRNAs using the same method as described in (E), except for the omission of 20 IVS tRNAs. The expected bands for mature tRNA are indicated by yellow arrowheads. Three independent experiments were conducted (See Supplementary Fig. 3), and representative data are shown.