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。它的重点是多蛋白介质复合物，它被认为是真核生物中几乎所有RNA聚合酶ii转录基因转录的关键辅激活因子。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其多种功能能力，Mediator被认为是转录信号最终处理的主要通道。一些中介亚基的异常表达也与包括癌症、心脏异常和神经变性在内的人类疾病有关。尽管它们有共同的进化起源，酵母和哺乳动物复合体却有很大的不同。与酵母介质的情况不同，遗传研究强调了重要的结构-功能关系，并为生化研究指明了道路，技术问题使得很难超越对30个亚基哺乳动物复合体的结构及其功能的粗略描述。尚未解决的问题涉及综合体的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，我们建议采用一种系统的方法来定义核心人类复合体的模块化结构（特别是当它涉及其基本功能时），并开发从各种生物来源中纯化完整复合体及其相互作用蛋白质的方法。为了实现这些目标，我们的目标是(i)重组地生成模块化中介复合体的最小泛函导数；（ii）培养针对特定中介表位的单克隆抗体，这将使与相互作用因子相关的天然复合物易于纯化。因此，这些工具的可用性有望为人类中介领域的下一阶段研究提供一个快速启动。

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.