Human mitochondrial transcription termination exhibits RNA polymerase independence and biased bipolarity in vitro

Human mitochondrial 16 S rRNA 3'-end formation requires a tridecamer template sequence and a trans-acting protein of approximately 34 kDa. This protein binds tightly to its target sequence and further analysis of the protein-DNA complex revealed that the DNA is bent. Either T3, T7, Escherichia coli, or yeast mitochondrial RNA polymerase produced transcripts mapping at this termination site. With these heterologous RNA polymerase, RNA 3'-end formation was detected only in the transcription polarity opposite that of mitochondrial rRNA synthesis; the efficiency of termination in the homologous human RNA polymerase system is approximately 2-fold greater in this same opposite polarity. These results suggested the possible importance of biased bipolar transcription termination in vivo. For wild-type mtDNA, the apparent relative efficiency of termination in vivo reflected the values determined in vitro. Examination of a pathogenic human mtDNA mutation known to result in impaired termination in vitro showed no significant differences in relative transcript abundances in vivo, despite a loss of in vitro termination efficiency in both directions. Recently, six additional mitochondrial disease-associated point mutations have been reported that cluster at the human mitochondrial transcription termination site. None of these resulted in significantly impaired transcription termination in vitro.