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

Figure 6.

Figure 6.

Mitochondrial promoters are recognized cooperatively by mtRNAP and TFB2M. (A) Mismatched nucleotides introduced into the NT DNA strand at positions -1 or -4 affect transcription efficiency. Transcription was performed using human proteins, and human LSP templates with single nucleotide mismatches at the position indicated. (B) Substitution of S. scrofa TFB2M with H.s. TFB2M compensates for the defects in the transcription of porcine LSP having mutations at positions -1 and -4. Transcription was performed using porcine mtRNAP and TFAM, and porcine (lanes 1–4) or human (lanes 5–8) TFB2M. LSP templates with single base-pair substitutions (porcine to human base) at the position indicated were used. Note the efficient transcript slippage observed on the -1A mutant template due to the generated stretch of three TMP bases in the template DNA strand (lanes 2 and 6). (C) Human TFB2M recognizes the dAMP base at the -1 position in the non-template strand. The native porcine LSP or its variants having all possible base-pair substitutions at position -1 were used in transcription assay with porcine TFAM, mtRNAP, and human TFB2M. (D) Human mtRNAP recognizes the -4 base in the template DNA strand. Human LSP template having base-pair substitutions at positions -1 and -4 (lane 1, template I) or a base pair substitution at position -1 and a mismatch at base -4 (lane 2, template II) were transcribed with human mtRNAP and TFAM, and porcine TFB2M. (E) Schematic illustration of promoter recognition by mtRNAP and TFB2M. MtRNAP recognizes the -3 and -4 bases in the template strand, while TFB2M recognizes the -1 and -4 bases in the non-template strand. (F, G) Porcine transcription machinery can recognize a modified human LSP. Transcription assays involved native human LSP (III), a porcinized human LSP with base-pair substitutions at positions -1, -3, and -4 (IV), native porcine LSP (V), and a modified (humanized) porcine LSP with base-pair substitutions at positions -1, -3 and -4 (VI). The promoter sequences near TSS are shown to the left (F). The left panel of the gel in (G) represents transcription reaction using human proteins, the right panel - transcription with porcine proteins.