DISTINCT ROLES FOR 2 PURIFIED FACTORS IN TRANSCRIPTION OF XENOPUS MITOCHONDRIAL-DNA

Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mi tochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtT FA revealed a high degree of sequence similarity to human and Saccharo myces cerevisiae mtTFA, xl-mtTFA was not required for basal transcript ion from a minimal mtDNA promoter, and this HMG box factor could not s ubstitute for the basal factor, which is therefore designated xl-mtTFB . An antibody directed against the N terminus of xl-mtTFA did not cros s-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprin ting experiments identified preferred binding sites for xl-mtTFA withi n the control region of mtDNA next to major mitochondrial promoters. W e show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcripti on in vitro by the basal transcription machinery, comprising mitochond rial RNA polymerase and xl-mtTFB.