Structure-Function Studies of the Modular Human Mediator Coactivator Complex

描述（由申请人提供）：本申请涉及广泛的挑战领域06：使能技术和特定的挑战主题06-GM-101：大分子复合物的结构分析。它侧重于多蛋白介体复合物，它已被牵连作为一个关键的辅激活转录基本上所有的RNA聚合酶II转录基因在真核生物。通过其与转录调节因子和RNA聚合酶机制相互作用的能力以及通过其多功能能力，介体被认为是转录信号最终加工的主要通道。几种介体亚基的异常表达也与人类疾病有关，包括癌症、心脏异常和神经变性。尽管酵母菌和哺乳动物复合体有着共同的进化起源，但它们却有着相当大的分歧。与酵母介体的情况不同，遗传学研究强调了重要的结构-功能关系，并为生化研究指明了方向，技术问题使得很难超越对30个亚基哺乳动物复合体及其功能的结构的粗略描述。尚未解决的问题涉及建筑群基本核心的定义以及在不同条件下影响其功能的其他因素的性质。在这里，我们建议采取一种系统的方法来定义的核心人类复合体的模块化架构（特别是因为它涉及到其基本功能），并开发方法来纯化完整的复合体及其相互作用的蛋白质从不同的生物来源。为了实现这些目标，我们的目标是（i）重组产生模块化介体复合物的最小功能衍生物;和（ii）产生针对限定的介体表位的单克隆抗体，其将允许与相互作用因子相关的天然复合物的容易纯化。因此，这些工具的可用性预计将为人类调解人领域的下一阶段研究提供一个快速启动。 公共卫生相关性：多亚基介导子已经成为参与蛋白质编码基因精确转录的最关键因子之一，其异常表达是许多人类疾病，特别是癌症的原因。尽管人类介体的几个单独的亚基直接涉及包括结肠直肠癌、心脏异常和神经变性在内的疾病，但人类介体的详细研究面临着由于其稀缺性、非凡的大小和组成的异质性而产生的重大技术挑战。通过直接解决这些挑战，该提案旨在揭示对人类介体组成的新见解，并通过产生重要的新的基于抗体的工具，它旨在为许多转录相关疾病的分子基础的预期未来工作奠定基础。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area 06: Enabling Technologies and specific Challenge Topic 06-GM-101: Structural Analysis of Macromolecular Complexes. It focuses on the multiprotein Mediator complex, which has been implicated as a critical coactivator for transcription of essentially all RNA polymerase II-transcribed genes in eukaryotes. Through its ability to interact with both transcriptional regulators and the RNA polymerase machinery as well as through its multiple functional capabilities, Mediator is believed to be the main channel through which transcriptional signals are ultimately processed. Aberrant expression of several Mediator subunits has also been implicated in human diseases that include cancer, heart abnormalities, and neural degeneration. Despite their common evolutionary origins, the yeast and mammalian complexes have diverged considerably. Unlike the situation for the yeast Mediator, where genetic studies have highlighted important structure-function relationships and led the way for biochemical studies, technical issues have made it difficult to go beyond a gross description of the architecture of the 30-subunit mammalian complex and its function. Unaddressed issues relate to definition of the essential core of the complex and to the nature of other factors that impinge on its function under different conditions. Here we propose to undertake a systematic approach both to define the modular architecture of the core human complex (especially as it pertains to its essential functions) and to develop methods to purify the intact complex and its interacting proteins from a diverse range of biological sources. Towards these goals we aim (i) to recombinantly generate a minimal functional derivative of the modular Mediator complex; and (ii) to raise monoclonal antibodies against defined Mediator epitopes that will allow facile purification of the natural complex in association with interacting factors. Availability of these tools is thus expected to provide a jump-start to the next phase of studies in the human Mediator field. PUBLIC HEALTH RELEVANCE: The multisubunit Mediator has emerged as one of the most critical factors involved in the precise transcription of protein-encoding genes, the aberrant expression of which is a cause of many human diseases, especially cancer. Although several individual subunits of the human Mediator have been directly implicated in diseases including colorectal cancer, heart abnormalities and neural degeneration, detailed studies of the human Mediator have faced significant technical challenges arising from its scarcity, extraordinary size and heterogeneity with respect to its composition. By directly addressing these challenges, this proposal aims to reveal new insights into the composition of the human Mediator, and, by generating important new antibody-based tools; it aims to lay the foundation for anticipated future work into the molecular bases of many transcription-related diseases.