FIGURE 1.

TFB2M but not TFB1M is required to stimulate human mitochondrial transcription initiation. A, sequences of LSP and HSP1 aligned at their transcription start sites. Shaded boxes indicate transcripts obtained in reaction having limited sets of the substrate NTPs. B, transcription of HSP1 and LSP in the presence of POLRMT, TFAM, and TFB2M. The reactions were carried out as described under “Experimental Procedures” using ApA primer and limited NTP mixtures, and the products were resolved by 20% PAGE containing 6 m urea. C, transcription of HSP1 and LSP in the presence of POLRMT, TFAM, and TFB1M. The reactions were carried out as described above. Note that the image is overexposed (6-fold) as compared with the experiment shown in panel B to demonstrate TFAM effect on transcription by POLRMT. D, transcription of LSP and HSP1 depends on TFAM stimulation. The reactions were carried out as described above using POLRMT, TFB2M (all 50 nm), and TFAM concentrations as indicated (0–200 nm). E, single-round transcription using catalytic autolabeling. Reactions containing POLRMT (150 nm), TFB2M (150 nm), and TFAM (0–500 nm) were performed as described under “Experimental Procedures” and resolved using 4–12% Bis-Tris NuPAGE.