Understanding the details of how genetic information is expressed from the separate mitochondrial genome requires a detailed description of the properties of the mitochondrial RNA polymerase. This nuclear-encoded enzyme is necessary and sufficient for the transcription of all mitochondrially encoded genes. Mitochondria from yeast to humans use a single-polypeptide catalytic RNA polymerase related to enzymes from bacteriophage. They also require separable transcription factors necessary for initiation at promoter sequences on the mitochondrial DNA template. It has recently become possible to work with highly purified, recombinant forms of the mitochondrial RNA polymerase subunits from yeast. This chapter describes detailed protocols for working in vitro with this purified enzyme in transcription reactions. These assays are critical for elucidating the nature of a mitochondrial promoter and for understanding how the mitochondrial RNA polymerase recognizes these DNA sequences and selectively initiates the transcription cycle, resulting in discrete transcripts.
