FIGURE 2.
Basal transcription system requires only TFAM, TFB2M, and two promoters, LSP and HSP1. A, MRPL12 does not stimulate mitochondrial transcription in vitro in the presence of the purified recombinant proteins. Reactions containing synthetic HSP1 template were performed as in Fig. 1B except that ATP, GTP, and CTP (all 0.3 mm) were used. B, MRPL12 does not stimulate transcription in the presence of mitochondrial extracts. For experiments with recombinant proteins (lanes 1–6), the individual transcription reaction mixtures contained POLRMT (400 fmol), TFB2M (400 fmol), TFAM (2.5 pmol), and the indicated mtDNA template (85 fmol). An S-100 mitochondrial lysate was used for transcription with the extracts (lanes 7–12). C, sequence of the HSP2 region. Shaded boxes indicate the 3′-terminal end of the tRNAPhe gene and the 5′-end of 12 S RNA gene. D, run-off transcription assay using promoter templates containing LSP1, HSP1, and HSP2. Transcription was carried out in the presence of TFAM, TFB2M, and POLRMT for 40 min at 35 °C using 0.5 mm substrate NTPs. E, run-off transcription assay using plasmid templates containing LSP and HSP1/HSP2. Transcription reactions were performed as indicated in panel B. HSP1/HSP2 template was obtained by linearization of a human mtDNA fragment at position 741. Transcription of HSP1 transcription generated a 181-nt product, but no 96-nt product expected for initiation at HSP2 was observed. LSP transcription using an mtDNA fragment corresponding to positions 1–477 generated run-off and prematurely terminated (PT) products. F, transcription is initiated from HSP1, but not from HSP2, in the presence of an S-100 mitochondrial lysate. A transcription reaction using HSP1/HSP2 template (lanes 1 and 2) described above was performed in the presence of the mitochondrial extracts supplemented with TFAM (1 pmol) and TFB2M (0.25 pmol). The positions of size markers (SM) are given to the left.