Mechanism of Transcription Initiation and Primer Generation at the Mitochondrial Origin of Replication, OriL

Azadeh Sarfallah, Thomas Jefferson University

Abstract

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. To address these mechanisms, we reconstituted OriL in vitro transcription and coupled transcription-replication assays. We found that unlike promoter-driven transcription, OriL transcription is independent of transcription factors and does not require the structural elements of mtRNAP involved in promoter recognition. However, the efficiency of transcription at OriL is significantly lower than the one at the light-strand promoter (LSP). We defined the OriL transcription start site and found that transcription initiation involves slippage of the nascent transcript, a process of repetitive addition of AMP residues to the transcript without translocation of polymerase relative to the DNA template. Our results indicate that the transcript slippage is required for initiation complex stability and its ability to generate RNA primer without translocation. Our data suggest a model of transcription replication, according to which the mitochondrial DNA polymerase gamma, PolG, pre-binds to the stem region of OriL, leaving the loop of the OriL hairpin available to mtRNAP binding. Upon generation of ~30 nt RNA primer, mtRNAP dissociates and the 3’ end of the RNA is taken over by PolG, which commences synthesis of the nascent DNA strand. Our data suggest the primosome assembly at OriL - a complex of mtRNAP and PolG - can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

Subject Area

Biochemistry|Molecular biology

Recommended Citation

Sarfallah, Azadeh, "Mechanism of Transcription Initiation and Primer Generation at the Mitochondrial Origin of Replication, OriL" (2021). ETD Collection for Thomas Jefferson University. AAI28719831.
https://jdc.jefferson.edu/dissertations/AAI28719831

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