New Paradigms for the molecular basis of RNA polymerase I transcription

项目摘要 真核RNA聚合酶I（Pol I）转录核糖体RNA，核糖体的关键组分。Pol I转录 占细胞中总RNA的大部分，其在人类细胞中的上调是癌症的标志， 其下调是几种发育障碍的标志。Pol I转录研究不足， 到波尔二世甚至波尔三世的转录。我们的初步工作表明， 以及作为本提案基础的第二和第三项政策。我们广泛的长期目标是确定 Pol I转录的机制及其失调如何导致癌症和发育障碍。那里 是我们理解的主要差距（1）Pol I转录的结构组织和架构 （2）Pol I起始因子与编码核糖体RNA的rDNA相互作用的机制; （3）Pol I转录因子在激活过程中的分子功能。第一个理由 这项工作的目的是确定Pol I转录的机制和调控将形成分子基础， 了解Pol I缺陷如何导致人类疾病。我们的中心假设是，Pol I因子使用 独特的机制进行转录和它们的结构和功能是不同的机制 控制Pol II和III转录。第二个基本原理是理解Pol I转录机制 在最基本和最根本的层面上，将转化为更好地理解第一次世界大战和第二次世界大战之间的联系， 和癌症的关系，从而产生新的癌症治疗策略。我们提出的研究将使用一个概念和 技术创新跨生物体和跨学科的方法， 酵母中的计算、分子、生物化学、遗传、基因组、蛋白质组和结构方法， 人体细胞在强有力的初步研究的指导下，我们将测试两个具体目标：（1）确定独特的 TATA结合蛋白（TBP）在Pol I转录中的“共激活剂”作用;（2）确定Pol I转录的机制 转录激活为了实现这些目标，我们将采用行之有效的互补方法 以鉴定和绘制其天然背景下的新型Pol I相互作用。我们将补充这些研究与结构 结合分子、遗传和生物化学功能测定进行建模，以鉴定Pol I因子功能 从酵母菌到人类都保存了下来拟议的研究是有意义的，因为它将导致详细的描述 的Pol I转录机制，并将提供一个概念框架，了解之间的联系， Pol I与人类疾病。最终，这项工作将为诊断，潜在的干预， 以及针对这些新的蛋白质-蛋白质和蛋白质-DNA相互作用的疗法的开发。

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.