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. 2014 Jan 6;42(6):3884–3893. doi: 10.1093/nar/gkt1356

Figure 1.

Figure 1.

TFAM makes direct interactions with mtRNAP. (A) TFAM is absolutely required for efficient transcription of both mtDNA promoters. In vitro transcription assay was performed with the nucleotide sets lacking CTP using PCR amplified templates with the HSP1 (lanes 1,2) or LSP (lanes 3,4) promoters. The gel image is overexposed to dramatize lack of transcription initiation on the LSP and trace activity (<0.5%) observed on the HSP1 in TFAM absence. (B) TFAM-mtRNAP interactions do not require TFB2M but depend on the DNA presence. The complexes were assembled using MBP-modified Cys217TFAM and 32P-labeled mtRNAP and TFB2M (where indicated) and UV-irradiated in the absence (lanes 1,2) or in the presence (lanes 3,4) of DNA. (C) Location of the residues probed in photo cross-linking experiments using MBP or pBpa (yellow spheres) on TFAM-DNA structure. (D) Scanning cross-linking of pBpa-containing TFAM and mtRNAP. The pre-initiation complexes were assembled using 32P-labeled mtRNAP (50 nM), 50 nM LSP and 50 nM TFAM having pBpa at the position indicated, UV irradiated and resolved in SDS-PAGE. Note that covalently linked polypeptides may migrate differently depending on the point of attachment. (E) TFAM does not cross-link to the heterologous mtRNAP. Pre-initiation complexes were assembled using MBP-modified Cys217TFAM and mtRNAP (lanes 1–3) or yeast mtRNAP (RPO41) (lanes 4,5) in the absence or presence of DNA (LSP for human mtRNAP and 14S promoter for RPO41), as indicated and UV-irradiated. Molecular weight markers are shown in lane 6. Note that molecular weight of RPO41 (155 kDa) is similar to that of TFAM-mtRNAP cross-link. (F) TFAM does not cross-link to TFB2M. Initiation complexes (150 nM) were assembled using mtRNAP, 32P-labeled TFB2M, MBP-modified TFAM and the LSP promoter. The grey arrow with an asterisk marks the expected position of the TFB2M-TFAM cross-linking species. (G) TFAM-mtRNAP interactions require DNA long enough to accommodate both proteins. Cross-linking was performed using Cys217MBP-TFAM and WT RNAP and synthetic double-stranded DNA having nonspecific sequence and the lengths indicated.