Figure 2.

Synthesis of mtDNA-encoded proteins by the reconstituted mammalian mitochondrial translation system. (A–C) Translation reactions (coupled transcription, aminoacylation, and translation reactions) were performed using the indicated template DNA and E. coli tRNA mix in the presence of [³⁵S] methionine. After the 120 min reaction, the samples were treated with RNase A and fractionated by 15% SDS-PAGE. The schematic of the mRNA produced from the template DNA during the reaction is shown below the panel. (A) Reactions with the standard template DNA; (B) reactions with the control template DNA, in which the initiation AUG codon in the standard template DNA is mutated to ACG and (C) reactions with the control template DNA, in which the λcI sequence in the standard template DNA is deleted. Full gel images are shown in Supplementary Figure S2. Another set of experiment corresponding to (C) is shown in Supplementary Figure S2C. White arrowheads indicate the relevant translation products, and asterisks denote unknown translation products. DHFR, E. coli dihydrofolate reductase; ND1–6 and ND4L, NADH dehydrogenase subunits; ATP6 and ATP8, ATP synthase subunits; CO1–3, cytochrome c oxidase subunits; CytB, cytochrome b. Molecular weights of DHFR and the mtDNA-encoded proteins, without the λcI sequence (kDa): DHFR(18), ATP6(24.8), ATP8(8), CO3(30), CO2(25.6), CO1(57), CytB(42.7), ND1(36), ND2(39), ND3(13), ND4L(11), ND4(52), ND5(67), ND6(18).