Conserved sequence box II directs transcription termination and primer formation in mitochondria

The human mitochondrial transcription machinery generates the RNA primers needed for initiation of heavy strand DNA synthesis. Most DNA replication events from the heavy strand origin are prematurely terminated, forming a persistent RNA-DNA hybrid, which remains annealed to the parental DNA strand. This triple-stranded structure is called the D-loop and encompasses the conserved sequence box II, a DNA element required for proper primer formation. We here use a purified recombinant mitochondrial transcription system and demonstrate that conserved sequence box II is a sequence-dependent transcription termination element in vitro. Transcription from the light strand promoter is prematurely terminated at positions 300-282 in the mitochondrial genome, which coincide with the major RNA-DNA transition points in the D-loop of human mitochondria. Based on our findings, we propose a model for primer formation at the origin of heavy strand DNA replication.