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：使能技术和特定挑战主题06-GM-101：大分子结构分析。它集中在多蛋白介体复合体上，它被认为是真核生物中几乎所有RNA聚合酶II转录的基因转录的关键辅助激活因子。通过与转录调控因子和RNA聚合酶机制的相互作用，以及通过其多功能功能，Mediator被认为是最终处理转录信号的主要渠道。一些介体亚单位的异常表达也与人类疾病有关，包括癌症、心脏异常和神经退化。尽管酵母和哺乳动物的复合体有着共同的进化起源，但它们之间的差异很大。与酵母介体的情况不同，遗传研究强调了重要的结构-功能关系，并引领了生化研究的道路，而技术问题使人们很难超越对这个由30个亚单位组成的哺乳动物复合体的结构及其功能的粗略描述。未解决的问题涉及复合体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核心人类复合体的模块结构(特别是当它与其基本功能有关时)，并开发从不同的生物来源中纯化完整的复合体及其相互作用的蛋白质的方法。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体的最小功能衍生物；以及(Ii)产生针对已定义的介体表位的单抗，这将允许与相互作用因子相关联的自然复合体的简便纯化。因此，这些工具的提供可望为人类调解人领域下一阶段的研究提供一个突破口。 公共卫生相关性：多亚单位介体已成为参与蛋白质编码基因精确转录的最关键因素之一，其异常表达是许多人类疾病，特别是癌症的原因之一。尽管人类介体的几个亚基直接与结直肠癌、心脏畸形和神经变性等疾病有关，但由于人类介体的稀缺性、巨大的体积和组成的异质性，对人类介体的详细研究面临着巨大的技术挑战。通过直接应对这些挑战，这项提议旨在揭示对人类介体的组成的新见解，并通过产生重要的新的基于抗体的工具；它旨在为预期的未来对许多转录相关疾病的分子基础的工作奠定基础。

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.