biostudies-literatureSarfallah AFoundation for the National Institutes of HealthNIGMS NIH HHSe107988https://www.ebi.ac.uk/biostudies/studies/S-EPMC8488568biostudies-literatureUnknown40(19)The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the priThe EMBO journalMechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.PMC8488568R35 GM131832Zamudio-Ochoa ASarfallah ATemiakov DAnikin MfalseMechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the pri2021-01-01T00:00:00Z2021 Oct2026-06-01T02:24:18.244Z2025-04-04T12:05:17.209ZS-EPMC84885683442345210.15252/embj.2021107988