Run-off in vitro transcription assays preformed using mtDNA templates with or without TFAM binding site variants.

A. A schematic map of the mtDNA templates used in the run-off in vitro transcription assay, with the TFAM-binding sites (striped rectangles), light-strand promoter (LSP, bent arrow), and the location of the 242 and 295 variants (arrows) indicated. B. A representative in vitro transcription reaction using templates containing the indicated mtDNA variants. Equal amounts of the linear mtDNA templates (ethidium bromide-stained at the bottom) were used in the in vitro transcription assay with a partially purified POLRMT (i.e. mtRNA polymerase) fraction from HeLa cells. The 223-nt full-length run-off transcript and a second major, shorter transcript of ∼160 nt (see Discussion) are indicated. Quantification of the 223-nt, full-length transcript and the ∼160-nt truncated product from multiple independent experiments is shown in C. and D., respectively. Both mutant templates showed a trend toward increased LSP transcription activity when either the full-length or shorter product was analyzed, but only in the case of shorter, ∼160 transcript from the haplogroup J template (C295T) was this difference statistically significant (p-value 0.034).