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

人类线粒体中的转录起始是由专用的线粒体RNA聚合酶（mtRNAP）在两种通用转录因子TFAM和TFB 2 M的辅助下进行的。为了启动转录，mtRNAP、TFAM和TFB 2 M在启动子DNA上组装，严重扭曲启动子，并熔化启动子DNA。然后mtRNAP开始合成前mRNA并逃离启动子。虽然线粒体转录机制的组成部分以及它们的结构是已知的，但对该过程的动力学知之甚少。实时单分子功能测定提供了对多步骤过程的动力学的独特见解，并且可以建立单个转录因子分子的结合/解离与随后的前体RNA合成之间的因果关系。最近，Revyakin实验室的博士生艾米丽蒂斯与美国Temiakov实验室合作，在单分子超分辨率荧光显微镜下重建了人类线粒体转录系统，并在完整的转录周期中解剖了mtRNAP和TFAM的协同动力学。然而，启动的最关键步骤-启动子选择和启动子熔化，据信是由TFB 2 M进行的-目前仍然是谜。在这篇博士论文中，Rory将制备一种活性荧光标记的人TFB 2 M，并进行二、三、四色单分子实验，以探讨TFB 2 M在启动子识别、启动子逃逸和线粒体转录中启动子方向性选择中的作用。我们的假设是，TFB 2 M起着类似于细菌大肠杆菌RNA聚合酶的σ因子的作用，通过瞬时进入mtRNAP前起始复合物，并在启动子逃逸后通过mtRNAP解离。我们进一步假设TFB 2 M的结合模式决定了线粒体基因组中“轻”DNA链或“重”DNA链转录方向的选择。

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.