12] Mitochondrial DNA transcription initiation and termination using mitochondrial lysates from cultured human cells

Abstract

Publisher Summary This chapter discusses mitochondrial DNA transcription initiation and termination using mitochondrial lysates from cultured human cells. In human mitochondria, the main initiation sites for transcription of the heavy (H) and light (L) strands of the DNA have been identified in vivo and in vitro , the discrete transcription products have been mapped, and their structural and metabolic properties have been characterized. One mitochondrial transcription factor—mtTFA—necessary for high levels of specific initiation of transcription at the L-strand promoter and rRNA-specific H-strand promoter, has also been identified and cloned. A different DNA-binding factor, mTERF—involved in termination of transcription at the 16 S rRNA/tRNA Leu(UUR) boundary—has been extensively characterized in the laboratory. On the other hand, nothing is known about the mtRNA polymerase itself and the probable existence of other factors involved in transcription initiation at the downstream, whole H-strand specific promoter. The chapter reports an adaptation of the mobility shift assay for the semiquantitative detection of mitochondrial transcription termination factor (mTERF) in the S-13 fraction from small amounts of cells, which allows an easy comparison of its relative content in different cell lines and under different growth conditions.