Molecular Mechanisms of Mitochondrial Transcription and Replication

线粒体转录和复制的分子机制

基本信息

批准号：
10620300
负责人：
Dmitry Temiakov
金额：
$ 53.35万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2024-05-31
项目状态：
已结题

项目摘要

! PROJECT SUMMARY Defects in mtDNA replication and expression result in myopathies, hearing and vision loss, and other devastating disorders. The key enzyme involved in mtDNA expression, mitochondrial RNA polymerase (mtRNAP) belongs to a family of single-subunit RNAPs that is distinct from the multi-subunit cellular RNAPs. Human mtDNA contains only two promoters that share limited sequence homology; the molecular basis for recognition of these promoters remains unclear. We will use the recently obtained structures of the initiation complex to guide biochemical experiments and probe the function of mtRNAP domains involved in promoter binding, recognition, and melting. Our studies will determine the role the initiation factors play in the mechanisms of promoter recognition. We will obtain the high-resolution structures of the pre-initiation and initially transcribing initiation complexes. In the absence of transcription initiation factors mtRNAP is capable to initiate transcription on a hairpin promoter at the origin of replication OriL and thus generates replication primers. We will elucidate how mtRNAP recognizes and binds this unusual promoter and what factors affect this process. The structure of OriL-IC will be determined. Studies of the structure and function of the mtRNAP and molecular mechanisms of transcription and replication are important for understanding the regulation of mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial functions and as a consequence, to treat mitochondria-associated diseases. Human cells differ in the levels of mtDNA they contain. Changes in mtDNA copy number occur during normal developmental processes but have also been reported during various pathological processes and aging. At present, the mechanisms responsible for regulation of mtDNA copy number are poorly understood, impeding our ability to use mitochondria as a therapeutic target. The replication machinery in human mitochondria includes DNA polymerase gamma, TWINKLE helicase, single-stranded DNA binding protein and mtRNAP, which generates replication primers. Our recent studies identified mitochondrial transcription elongation factor, TEFM, as a major component of a molecular switch between replication and transcription in mitochondria that would allow the corresponding machineries to avoid the detrimental consequences of head-on collisions. How this switch is regulated in cells in response to different conditions is not understood. This research with determine molecular mechanism of TEFM action by biochemical and structural approaches and how changes in TEFM expression affect mtDNA copy number and mitochondrial transcription in cells during normal and stress conditions, and during cell differentiation. To elucidate the underlying mechanisms that control the set level of mtDNA, the interplay between the TEFM-dependent switch and the switch that operates at the D-loop region of mtDNA and regulates utilization of the replication intermediate, the 7S DNA, will be investigated.

呢项目摘要 mtDNA复制和表达缺陷导致肌病，听力和视力丧失以及其他毁灭性疾病。参与mtDNA表达的关键酶，线粒体RNA聚合酶（MTRNAP）属于一个与多亚基细胞RNAP不同的单人物RNAP家族。人mtDNA仅包含两个具有有限序列同源性的启动子。分子基础对这些启动子的认识尚不清楚。我们将使用最近获得的启动结构复合物指导生化实验并探测参与启动子的MTRNAP结构域的功能绑定，识别和融化。我们的研究将确定起始因素在启动子识别机制。我们将获得预先放电的高分辨率结构最初转录启动复合物。在没有转录引发因子的情况下，MTRNAP能够在复制olil的起源上启动发夹启动子的转录，从而产生复制底漆。我们将阐明MTRNAP如何识别和束缚这种异常的启动子以及哪些因素影响这个过程。将确定Oril-IC的结构。 MTRNAP的结构和功能的研究转录和复制的分子机制对于理解调节很重要线粒体基因组表达。反过来，这将决定我们影响各种线粒体的能力因此，功能和治疗线粒体相关疾病的功能。人类细胞在其中包含的mtDNA水平上有所不同。正常情况下，mtDNA拷贝数的变化发生发育过程，但在各种病理过程和衰老中也有报道。在目前，负责MTDNA拷贝数调节的机制知之甚少，阻碍了我们使用线粒体作为治疗靶标的能力。人线粒体中的复制机制包括DNA聚合酶伽马，闪烁解旋酶，单链DNA结合蛋白和MTRNAP，生成复制引物。我们最近的研究确定了线粒体转录伸长因子， TEFM，作为分子切换的主要组成部分，在线粒体中的复制与转录之间将允许相应的机械避免正面碰撞的有害后果。如何该开关在响应不同条件的细胞中受到调节。这项研究与通过生化和结构方法确定TEFM作用的分子机制，以及如何变化在TEFM中，在正常和压力条件和细胞分化期间。阐明控制集合的基本机制 mtDNA的水平，依赖TEFM的开关与在D环处运行的开关之间的相互作用将研究mtDNA区域并调节复制中间体7S DNA的利用。