Single-Molecule Dynamics of the Human Mitochondrial RNA Polymerase Preinitiation Complex

Abstract

Mitochondrial mRNA transcription requires assembly of the preinitiation complex (PIC), which contains promoter DNA, mitochondrial RNA polymerase (mtRNAP), and the two General Transcription Factors (GTFs), TFAM and TF2BM. After TFAM-initiated DNA bending and the assembly of the PIC, mtRNAP, with the help of TFB2M, unwinds the promoter DNA, begins RNA synthesis, escapes the promoter, and begins elongation of the nascent RNA. The existence of structural models for mtRNAP, the GTFs, and even PIC intermdiates do not fully explain the order of PIC assembly, the rate-limiting steps that must be overcome to initiate transcription and the fates of residual GTFs after mtRNAP escape. In order to investigate these questions, we visualised the initiation of mtRNAP in vitro using Co-localization Single Molecule Spectroscopy (CoSMoS) in real time, at a one second time resolution. Further to this, we dynamically captured the entry of TFAM and mtRNAP into single PICs, mtRNAP promoter escape, mtRNAP elongation, and mtRNAP re-initiation. By super-resolving the dynamic positions of mtRNAP and TFAM on the DNA during initiation, we have indicated that it is feasible to use single-molecule, real-time, structural analysis of the formation of the PIC by mitochondrial transcription components.