FIGURE 1.

Human mitochondrial transcription machinery produced in E. coli supports promoter-dependent transcription on established plasmid-based templates in vitro. a, recombinant human mitochondrial transcription proteins used in this study. Proteins (5 μg) were resolved by SDS-PAGE on a 10% gel and visualized by Coomassie staining. b, plasmid-based templates for in vitro transcription. Schematic of pUC18-LSP, pUC18-HSP1, and pUC18-LSP-HSP1 plasmids used in this study (21). The position of unique restriction sites and the sizes (in nucleotides, nt) of the corresponding run-off transcripts are indicated. A 120-nt transcript will be produced from LSP regardless of the restriction enzyme used because of premature termination caused by the conserved sequence block II (CSBII). c, transcription assays. Reactions were performed by combining h-mtTFA (100 nm) and h-mtTFB2 (20 nm) with linearized plasmid DNA (4 nm) in reaction buffer containing NTP mix (400 μm ATP, 150 μm CTP, 150 μm GTP, 10 μm UTP, and 0.2 μCi/μl [α-³²P]UTP) at 32 °C, initiated by addition of h-mtRNAP (20 nm), and quenched after 30 min by addition of stop buffer. Products were resolved by denaturing PAGE on 5% gels and visualized by phosphorimaging. The size of run-off transcripts produced from the different linearized plasmid templates is indicated. The size of selected bands from a 10-bp DNA ladder (M) is indicated as a reference.