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有助于许多其他重要功能的重要细胞器。线粒体含有它们自己的DNA（mtDNA），其编码细胞器功能所必需的基因的子集。这些基因通过拟南芥特异性转录和翻译机制表达。线粒体基因表达的失调已被越来越多地认为是衰老和人类疾病如帕金森病、高血压、糖尿病和癌症的重要贡献者。值得注意的是，虽然主要的线粒体DNA调控元件和人类线粒体DNA表达所需的基本机制已经确定，许多重要的问题，在哺乳动物线粒体转录的机制和调控仍然存在。在这里，我们提出了一系列的生化和遗传实验，首次调查人类线粒体转录复合物的动态及其与线粒体翻译机制的相互作用。在本计划的目标1中，将鉴定和分析与这三种mtDNA启动子相互作用的转录因子。基础转录因子与线粒体DNA的相互作用也将通过染色质免疫沉淀方法进行研究。在目标2中，将讨论人类线粒体中转录和翻译机制之间的联系。转录复合物的亲和纯化，然后进行蛋白质印迹和质谱分析将用于研究线粒体中的转录-翻译偶联。最后，在目标3中，我们将研究MRPL 12的功能，MRPL 12是一种独特的线粒体核糖体蛋白，与线粒体RNA聚合酶相互作用并调节线粒体基因的转录。将分析MRPL 12过表达对线粒体生物发生和功能的影响。MRPL 12与转录机器相互作用的功能意义也将被检查。本提案中描述的实验的完成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础线粒体生物学的理解。此外，这项研究将对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的研究产生重大影响。) 公共卫生相关性：线粒体是含有其自身DNA（mtDNA）的重要细胞器，其失调与人类疾病和衰老有关。虽然线粒体DNA表达的调节被认为是线粒体病理学的潜在疗法，但这种方法受到我们对该过程如何调节的不完整知识的限制。本提案中对人类线粒体转录复合体的详细分析将大大增加我们对线粒体基因表达的理解，使我们更接近这一目标。

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.