Single-molecule dynamics of transcription initiation by the human mitochondrial RNA polymerase

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Transcription initiation in human mitochondria is carried out by a dedicated mitochondrial RNA polymerase (mtRNAP) assisted by two general transcription factors, termed TFAM and TFB2M. To initiate transcription, mtRNAP, TFAM, and TFB2M assemble on promoter DNA, severely distort the promoter, and melt the promoter DNA. The mtRNAP then begins premRNA synthesis and escapes the promoter. Although the components of the mitochondrial transcription machinery, as well as their structures, are known, the dynamics of the process are poorly understood. Real-time single-molecule functional assays provide a unique insight into dynamics of multi-step processes, and can establish the cause-and-effect relationships between binding/dissociation of individual transcription factor molecules and subsequent preRNA synthesis. Most recently, Emily Teece, a PhD student at the Revyakin lab, in collaboration with the Temiakov lab in the USA, has reconstituted the human mitochondrial transcription system under a single-molecule super-resolution fluorescence microscope, and dissected the concerted dynamics of mtRNAP and TFAM in full transcription cycles. However, the most critical steps of initiation - promoter selection and promoter melting, believed to be carried out by TFB2M - currently remain enigmatic. In this PhD thesis, Rory will prepare an active fluorescently labelled version of human TFB2M, and carry out two- three, and four-colour single molecule experiments that will probe the role of TFB2M in promoter recognition, promoter escape, and the selection of promoter directionality in mitochondrial transcription. Our hypothesis is that TFB2M plays a role similar to the sigma factor of the bacterial E coli RNA polymerase, by transiently entering the mtRNAP pre-initiation complex, and dissociating upon promoter escape by mtRNAP. We further hypothesize that the mode of binding of TFB2M determines the selection of directionality of transcription either from the 'light' DNA strand, or the 'heavy' DNA strand in the mitochondrial genome.