Past studies of mitochondrial transcription used chimeric templates in which only the transcription-control region of mtDNA was cloned into the plasmid pUC. We discovered sequences outside of the region essential for maximal transcriptional output. We characterized biochemical and biophysical differences between transcription on chimeric and authentic mtDNA templates by using atomic force microscopy and transcription assays. We also began to investigate how nuclear transcription factors interact with these templates.