Revealing the mechanism of directional transcription termination at the single molecule level for the human mitochondrial transcription complex

转录终止是产生功能性转录组（如基因组编码的全长RNA）和循环基因末端转录RNAP的必要细胞过程。然而，转录终止是一项困难的任务，因为一旦延伸-细胞RNA聚合酶（RNAP）与DNA形成非常稳定的复合物，能够合成数千个碱基长的RNA。因此，细胞已经发展出几种策略来有效地终止和循环转录基因末端的RNAP。其中一种策略使用结合到特定终止位点的蛋白质终止因子，如Pol I和人类线粒体RNAP （mtRNAP）。有趣的是，Pol I和mtRNAP是定向终止的，即只有其中一条链的转录在与终止因子碰撞时导致终止。尽管它们很重要，但这些终止因子感知Pol I和mtRNAP转录方向的机制尚不清楚。作为真核细胞的动力源，线粒体对细胞稳态至关重要，线粒体基因组的表达和维持至关重要。在这里，我打算揭示终止因子MTERF1如何仅从一个方向感知和终止转录mtRNAP。为了揭示这一机制，我将使用单分子生物物理学技术，因为它们独特地适合于询问本质上不均匀的酶促反应，如转录，并使生物分子的空间和力操纵成为可能。本研究结果将提供由终止因子诱导的定向转录终止的完整机械化学，并为未来人类线粒体转录调控的研究铺平道路。

Transcription termination is an essential cellular process to generate a functional transcriptome, e.g. full length RNA’s as encoded in the genome, and to recycle transcribing RNAP at the end of genes. Transcription termination is a difficult task though, as – once in elongation – cellular RNA polymerases (RNAP) form a very stable complex with DNA, capable of synthesizing kilobases long RNA’s. Therefore cells have developed several strategies to efficiently terminate and recycle RNAP’s at the end of transcribed genes. One of these strategies uses a protein termination factor bound to a specific termination site, as for Pol I and the human mitochondrial RNAP (mtRNAP). Interestingly, Pol I and mtRNAP are terminated directionally, i.e. only the transcription of one of the strands leads to termination upon collision with the termination factor. Despite their importance, the mechanism by which these termination factors sense the direction of transcribing Pol I and mtRNAP is unknown. As being the powerhouse of the eukaryotic cell, mitochondria are therefore essential for cell homeostasis, and the expression and maintenance of mitochondrial genome is of central importance. Here, I propose to uncover how the termination factor MTERF1 senses and terminates transcribing mtRNAP from only one direction. To reveal this mechanism, I will use single molecule biophysics techniques, as they are uniquely fitted to interrogate intrinsically heterogeneous enzymatic reactions, such as transcription, and enable the spatial and force manipulation of biomolecules. The findings of the present proposal will provide the complete mechanochemistry of directional transcription termination induced by a termination factor and pave the way for future studies of human mitochondria transcription regulation.