Figure 5.
Promoter recognition by human mtRNAP. (A) The -3 and -4 bases in the porcine promoter are important for promoter recognition. Transcription assays were performed using porcine proteins and native promoter (lanes 1 and 5) or promoter variants having substitutions at the -3 and -4 positions (Lanes 2–4, 6–8). (B) Schematic drawing illustrating the non-conserved residues in the specificity loop and the G helix in human (black) and porcine (blue) mtRNAP implicated in recognition of the bases -3 and -4 in the template strand of promoter DNA. (C) Human mtRNAP variants having substitutions in the G-helix and the specificity loop. The substitutions were made to match the corresponding residues found in porcine mtRNAP. (D) The G helix and the SP loop of mtRNAP recognize the -3 base in LSP. Transcription assay was performed using the native, ‘-3T’ or ‘-4A’ human LSP with the WT or mtRNAP mutants as indicated. (E) The pentamutant mtRNAP specifically recognizes the ‘-3T’ LSP. Transcription assays were performed using the native or mutant LSP, in which the -3 base was changed to all possible variants.
