Figure 2.

Human mitochondrial RNAP (hmRNAP) incorporates ADP analogs in vitro. A partial sequence of the light strand promoter (LSP) is shown, with the initially transcribed sequence underlined. For the assay, 50 nM TFAM, 50 nM hmRNAP, 50 nM TFB2M (purified as described in [23]) were combined with 50 nM of linear DNA fragment containing LSP promoter (positions -70 to +50) in 10 µl of transcription buffer (40 mM Tris pH 8.0, 10 mM MgCl₂, 10 mM DTT), then ATP or ADP analogs were added to the final concentration of 1mM. Transcription was initiated by the addition of 10 mM MgCl₂, 50 µM ATP, 300 µM GTP, 10µM [α³²P]-UTP, 25 Ci/mmol (Hartmann Analytic). After 30 min incubation at 37°C, 500 nM NudC was added to half of the reactions and incubated for additional 15 minutes at 37°C. Transcripts modified with NAD⁺, NADH and DP-CoA (but not ATP or FAD) were susceptible to NudC (lanes 5,9,11) judging from increased mobility of the products. Reactions were stopped by the addition of formamide-containing loading buffer. Products were separated on denaturing polyacrylamide gels (20% acrylamide, 3% bis-acrylamide, 6M urea, 1xTBE), revealed by PhosphorImaging (GE Healthcare), and analysed using ImageQuant software (GE Healthcare).