Analysis of Mammalian Mitochondrial Transcription Machinery

描述(申请人提供)：线粒体是重要的细胞器，提供大量细胞能量，并对许多其他重要功能作出贡献。线粒体包含它们自己的DNA(MtDNA)，编码细胞器功能所需的一组基因。这些基因通过线粒体特异的转录和翻译机制表达。线粒体基因表达的失调越来越被认为是导致衰老和帕金森氏症、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，虽然人类线粒体DNA表达所需的主要线粒体DNA调控元件和基础机制已经被确定，但关于哺乳动物线粒体转录的机制和调控的许多重要问题仍然存在。在这里，我们提出了一系列的生化和遗传学实验，以首次研究人类线粒体转录复合体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，将讨论人类线粒体转录和翻译机制之间的联系。转录复合体的亲和纯化、Western blotting和质谱学将用于研究线粒体转录-翻译偶联。最后，在目标3中，我们将研究MRPL12的功能，它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，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。还将研究MRPL12与转录机制相互作用的功能意义。这项建议中描述的实验的完成将为线粒体转录提供根本的新见解，并将极大地增加我们对基本线粒体生物学的理解。此外，本研究将对基于调控线粒体基因表达和线粒体DNA复制的潜在治疗策略的寻找产生重大影响。) 与公共健康相关：线粒体是包含其自身DNA(MtDNA)的基本细胞器，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一种潜在的线粒体病理学治疗方法，但这种方法受到我们对这一过程如何调控的不完全了解的限制。这项建议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，并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.