New Paradigms for the molecular basis of RNA polymerase I transcription

项目摘要 真核细胞RNA聚合酶I(POL I)转录核糖体RNA，核糖体是核糖体的重要组成部分。POL I转录 占细胞总RNA的大部分，它在人类细胞中的上调是癌症的标志，而 它的下调是几种发育障碍的标志。与Pol I转录相比，对其研究不足 到Pol II甚至Pol III的转录。我们的初步工作表明，Pol I和Pol III之间存在根本差异 以及波尔II和波尔III，它们是这项提议的基础。我们广泛的长期目标是确定分子 Pol I转录的机制及其失调如何导致癌症和发育障碍。那里 是我们在理解(1)POL I转录的结构组织和体系结构方面的主要差距 (2)Pol I启动因子与编码核糖体RNA的rDNA相互作用的机制； 以及(3)几个关键的Pol I转录因子在激活过程中的分子功能。第一个理由 因为这项工作是确定Pol I转录的机制和调控将形成分子基础 了解POLI缺陷是如何导致人类疾病的。我们的中心假设是POL I因子使用 进行转录的独特机制和它们的结构和功能不同于机制 管理Pol II和III的转录。第二个基本原理是理解Pol I转录机制 在最基本和最基本的层面上将转化为更好地理解Pol I之间的联系 和癌症，导致新的癌症治疗策略。我们提议的研究将在概念上使用 技术创新的跨组织和跨学科的方法，利用生物信息学、 酵母菌的计算、分子、生化、遗传、基因组、蛋白质和结构方法 人类细胞。在强有力的前期研究的指导下，我们将测试两个具体目标：(1)确定唯一的 TBP在Pol I转录中的“共激活”作用，以及(2)确定Pol I的机制 转录激活。为了实现这些目标，我们将采用行之有效和相辅相成的方法 识别和映射Pol I在其母语环境中的新的相互作用。我们将与这些研究相辅相成的是结构 结合分子、遗传和生化功能分析进行建模以确定Pol I因子的功能 从酵母菌保存到人类。拟议的研究具有重要意义，因为它将导致详细的描述 并将为理解Pol I转录机制之间的联系提供一个概念框架 Pol I和人类疾病。最终，这项工作将阐明诊断、潜在干预、 以及针对这些新的蛋白质-蛋白质和蛋白质-DNA相互作用的治疗方法的开发。

Project Summary Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal RNA, a key component of ribosomes. Pol I transcription accounts for the majority of the total RNA in cells, and its upregulation in human cells is a hallmark of cancer while its downregulation is a hallmark of several developmental disorders. Pol I transcription is understudied compared to transcription by Pol II and even Pol III. Our preliminary work suggests fundamental differences between Pol I and Pols II and III that are the basis for this proposal. Our broad long-term objectives are to determine the molecular mechanism of Pol I transcription and how its dysregulation leads to cancer and developmental disorders. There are major gaps in our understanding of (1) the structural organization and architecture of Pol I transcription complexes; (2) the mechanism for how Pol I initiation factors interact with rDNA, which encodes ribosomal RNA; and (3) the molecular function of several key Pol I transcription factors in the activation process. The first rationale for this work is that determining the mechanism and regulation of Pol I transcription will form the molecular basis for understanding how Pol I defects lead to human disease. Our central hypothesis is that Pol I factors use a unique mechanism to carry out transcription and their structure and function is different from the mechanisms governing Pol II and III transcription. The second rationale is that understanding the Pol I transcription mechanism at the most basic and fundamental levels will translate to a better understanding of the connection between Pol I and cancer, leading to new cancer therapeutic strategies. Our proposed research will use a conceptually and technically innovative cross-organismal and interdisciplinary approach that employs a combination of bioinformatic, computational, molecular, biochemical, genetic, genomic, proteomic, and structural methods in the yeast and human cells. Guided by strong preliminary studies, we will test two specific aims: (1) Determine the unique “coactivator” role of TATA-binding protein (TBP) in Pol I transcription, and (2) Determine the mechanism of Pol I transcription activation. To accomplish these aims, we will use well-established and complementary approaches to identify and map novel Pol I interactions in their native context. We will complement these studies with structural modeling in combination with molecular, genetic, and biochemical functional assays to identify Pol I factor functions conserved from yeast to humans. The proposed research is significant because it will lead to a detailed description of the Pol I transcription mechanism and will provide a conceptual framework for understanding the link between Pol I and human disease. Ultimately, this work will illuminate new avenues for diagnosis, potential interventions, and the development of therapies targeting these novel protein-protein and protein-DNA interactions.