Analysis of Mammalian Mitochondrial Transcription Machinery

描述（由申请人提供）：线粒体是提供大量细胞能量并参与许多其他重要功能的基本细胞器。线粒体含有自己的DNA (mtDNA)，其编码细胞器功能所必需的基因子集。这些基因通过线粒体特异性转录和翻译机制表达。线粒体基因表达失调已被越来越多地认为是衰老和帕金森病、高血压、糖尿病和癌症等人类疾病的重要因素。值得注意的是，虽然已经确定了人类线粒体DNA表达所需的主要mtDNA调控元件和基础机制，但关于哺乳动物线粒体转录的机制和调控仍然存在许多重要问题。在这里，我们提出了一系列的生化和遗传学实验，首次研究人类线粒体转录复合物的动力学及其与线粒体翻译机制的相互作用。在本提案的目标1中，将鉴定和分析与三个mtDNA启动子相互作用的转录因子。基底转录因子与mtDNA的相互作用也将通过染色质免疫沉淀法进行研究。在Aim 2中，将讨论人类线粒体中转录和翻译机制之间的联系。转录复合物亲和纯化，然后进行Western blotting和质谱分析，将用于研究线粒体中的转录-翻译偶联。最后，在Aim 3中，我们将研究MRPL12的功能，MRPL12是一种独特的线粒体核糖体蛋白，可与线粒体RNA聚合酶相互作用并调节线粒体基因的转录。我们将分析MRPL12过表达对线粒体生物发生和功能的影响。我们还将研究MRPL12与转录机制相互作用的功能意义。完成本提案中描述的实验将为线粒体转录提供基本的新见解，并将大大增加我们对基本线粒体生物学的理解。此外，这项研究将对寻找基于调节线粒体基因表达和线粒体DNA复制的潜在治疗策略产生重大影响。)

DESCRIPTION (provided by applicant): Mitochondria are essential organelles that provide a bulk of cellular energy and contribute to many other important functions. Mitochondria contain their own DNA (mtDNA) which encodes a subset of genes necessary for the function of the organelle. These genes are expressed by mitochondria-specific transcription and translation machineries. Dysregulation of mitochondrial gene expression has been increasingly recognized as an important contributor to aging and to human diseases such as Parkinson's disease, hypertension, diabetes, and cancer. Notably, while major mtDNA regulatory elements and basal machinery required for human mitochondrial DNA expression has been identified, many important questions about the mechanism and regulation of transcription in mammalian mitochondria remain. Here, we propose a series of biochemical and genetic experiments to investigate for the first time the dynamics of human mitochondrial transcription complexes and their interaction with mitochondrial translation machinery. In Aim 1 of this proposal, transcription factors interacting with the three mtDNA promoters will be identified and analyzed. Interaction of basal transcription factors with mtDNA will also be investigated via chromatin immunoprecipitation approach. In Aim 2, connections between transcription and translation machineries in human mitochondria will be addressed. Affinity purification of transcription complexes followed by Western blotting and mass-spectrometry will be used to study transcription-translation coupling in mitochondria. Finally, in Aim 3, we will investigate the function of MRPL12, a unique mitochondrial ribosomal protein that interacts with mitochondrial RNA polymerase and modulates transcription of mitochondrial genes. Effects of MRPL12 overexpression on mitochondrial biogenesis and function will be analyzed. Functional significance of MRPL12 interaction with transcription machinery will also be examined. Completion of the experiments described in this proposal will provide fundamental new insight into mitochondrial transcription and will greatly increase our understanding of basic mitochondrial biology. Moreover, this study will have a large impact on the search for potential therapeutic strategies based on modulating mitochondrial gene expression and mitochondrial DNA replication. ) PUBLIC HEALTH RELEVANCE: Mitochondria are essential organelles that contain their own DNA (mtDNA), dysregulation of which is implicated in human disease and aging. While modulation of mtDNA expression is recognized as a potential therapy for mitochondrial pathology, this approach is limited by our incomplete knowledge of how this process is regulated. The detailed analysis of human mitochondrial transcription complexes in this proposal will greatly increase our understanding of mitochondrial gene expression and bring us closer to this goal.