Single-molecule dynamics of human mitochondrial transcription

Mitochondria are the major source of energy production in the eukaryotic cell, and mitochondrial transcription is often dysregulated in cancers, ageing, and neurodegenerative diseases. Dissection of mitochondrial transcription at singlemolecule resolution - from initiation to escape to elongation - can provide new insights into mitochondrial transcriptional control and provide new methods to the field of real-time single-molecule spectroscopy. I have developed the first singlemolecule system to dissect transcription by the human mitochondrial RNA polymerase (RNAP), directly visualizing interactions between human mtRNAP and transcription factors TFAM, TFB2M, and TEFM, engaged in initiation and elongation from the Light Stand Promoter (LSP). My work revealed that mtRNAP preinitiation complexes assemble sequentially from monomeric components. TFAM binds to LSP first and recruits mtRNAP, which then recruits FB2M. Upon mtRNAP escape, TFB2M dissociates and immediately gets replaced by TEFM. I have measured all the characteristic binding and dissociation rates for all of the proteins involved, and have identified two key limiting steps in mtRNAP initiation: (i) mtRNAP recruitment to LSP is a slow process (kon ~= 1.1x105 M-1 s-1), consistent with TFAM finding and bending promoter DNA before recruiting mtRNAP; (ii) TFB2M makes several attempts to bind to mtRNAP and to, presumably, melt the promoter - at an overall initiation rate coefficient ~= 2.3x106 M-1 s-1. My measurements provide benchmarks for future analyses of mtRNAP transcription in live cells. In addition, methods and sensors developed will benefit researchers focused on long-term single-molecule imaging of functional molecular interactions in real-time.