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. 2022 Feb 22;50(5):2765–2781. doi: 10.1093/nar/gkac103

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Reconstitution and characterization of the porcine in vitro mitochondrial transcription system. (A Sequence alignment of the Sus scrofa promoters. TSS (+1) is indicated in blue. The sequence of human LSP is shown aligned to the porcine promoters based on the TSS location. Identical bases between the three promoters from bases -15 to + 5 are highlighted in yellow. (B, C) Transcription initiation by Sus scrofa mtRNAP is specific and critically depends upon TFAM and TFB2M. Transcription assays were performed using the PCR-amplified templates containing porcine LSP (B) or HSP (C). The reactions contained S. scrofa mtRNAP, TFAM, and TFB2M, as indicated. Products of transcription reaction involving human LSP (21 and 22 nt) were used as size markers (M). (D) Porcine initiation complex does not recognize human promoters. Transcription assays using human (H) or porcine (S) initiation complex proteins (mtRNAP, TFAM, and TFB2M) were performed using human LSP (left panel) or HSP (right panel). The blue and black font of the labels distinguishes the porcine and human proteins/sequences used. (E) Human transcription machinery does not recognize the porcine promoters. Transcription assays using human (H) or porcine (S) initiation complex proteins (mtRNAP, TFAM, and TFB2M) were performed using porcine LSP (left panel) or HSP (right panel). (F) Porcine TFAM can substitute for human TFAM during transcription initiation at LSP. Transcription assays were performed using human LSP, human mtRNAP, and TFB2M, and increasing concentrations of human (lanes 2–6) or porcine (lanes 7 -11) TFAM. (G) Close-up view showing the TFAM-mtRNAP interactions at LSP. Hydrogen bonds between the conserved residues are indicated. (H) Sequence conservation in the region of TFAM that interacts with mtRNAP. Identical residues are highlighted in yellow. (I) Porcine TFB2M cannot substitute for human TFB2M during transcription initiation at LSP. Transcription assays were performed using human LSP, human mtRNAP, and TFAM, and increasing concentrations of human (lanes 2 and 3) or porcine (lanes 4–7) TFB2M. (J) Human TFB2M has reduced activity when used with porcine LSP. Transcription assays were performed using porcine LSP, porcine mtRNAP, and TFAM, and increasing concentrations of porcine (lanes 2 and 3) or human (lanes 4 -7) TFB2M. (K) Human and porcine mtRNAPs are selective towards their corresponding promoters. Left panel. Transcription assays were performed using human LSP, TFB2M, TFAM, and human (lane 1) or porcine (lane 2) mtRNAP. Right panel. Transcription assays were performed using porcine LSP, TFB2M, TFAM, and human (lane 3) or porcine (lane 4) mtRNAP.