FIG. 5.

Recombinant human CGI-75 protein activates transcription from the human mitochondrial LSP. Mitochondrial transcription was assayed using an in vitro runoff transcription assay that measures specific transcription initiation from the human LSP. The resulting product is a discrete radio-labeled RNA transcript (indicated by an arrowhead) that begins at the LSP and extends to the end of the linear DNA template (10). Protein components present (+) or absent (−) in each reaction are indicated at the top of the figure. All reactions contained 5 μl of an mtTFA-dependent mitochondrial extract containing human mtRNA polymerase (HeLa mt extract). Where indicated, 2 μl of gel-purified and renatured h-mtTFA was added to the reaction mixture. Reaction mixtures contained recombinant human CGI-75 protein in the following amounts: lane 2, 20 ng; lane 4, 20 ng; lane 5, 16 ng; lane 6, 12 ng. The fact that decreasing amounts of CGI-75 were added in the last three reaction mixtures (lanes 4 to 6) is indicated at the bottom of the figure by the decreasing slope of the triangle from left to right.