Nucleic acid sequence determinants of transcriptional pausing by human mitochondrial RNA polymerase (POLRMT)

Transcription by RNA polymerase (RNAP) lies at the heart of gene expression in all organisms. The speed with which RNAPs produce the RNA is tuned in part by the signals in the transcribed nucleic-acid sequences, which temporarily arrange RNAPs into a paused conformation unable to extend the RNA. In turn, the altered transcription kinetics determines the three-dimensional shape into which RNA ultimately folds, dictates the chromatin state, and promotes or inhibits co-transcriptional events. While pause sequence determinants have been characterized for multi-subunit RNAPs in bacteria and the eukaryotic nuclei, this information is lacking for the single-subunit RNAP of human mitochondria, POLRMT. Here, we developed a robust nucleic-acid scaffold system to reconstitute POLRMT transcription in vitro and identified multiple transcriptional pause sites on the human mitochondrial genomic sequence (mtDNA). Using one of the pause sequences as a representative, we performed a suite of mutational studies to pinpoint the nucleic-acid elements that enhance, weaken, or completely abolish POLRMT pausing. Finally, a search of the human mtDNA for the pause motif revealed multiple predicted pause sites, with potential roles in mitochondrial co-transcriptional processes.