Control of RNA polymerase I transcription initiation and elongation

描述（由申请人提供）： 核糖体合成与细胞生长和增殖的速率直接相关。核糖体DNA（rDNA）通过RNA聚合酶I（Pol I）的转录是核糖体组装中的第一个限速步骤。在快速生长的细胞中，Pol I的转录占所有细胞转录的60%以上。该项目的总体假设是Pol I已经进化出支持有效、稳健活性的若干独特特征，并且这些特征是选择性抑制核糖体合成的逻辑靶标。为了验证这一假设并扩大对真核细胞中核糖体合成的理解，本项目将集中在rDNA转录的三个关键方面：1）Pol I的酶特性2）控制Pol I的反式作用因子3）rDNA中影响转录延伸和rRNA加工的序列。 Pol I已经进化出适合其细胞作用的酶特性。例如，最近的数据表明， Pol I与Pol II转录延伸中的限速步骤可能不同。这些差异可以作为选择性抑制核糖体生物合成的靶点。该项目将采用快速混合动力学测量和建模得到的数据集定量描述Pol I转录延伸。进一步的研究将确定转录的基本步骤 延伸受RNA聚合酶的特定亚基或结构域的影响。 Pol I转录需要反式作用因子来支持体内观察到的转录速率。为了检验这一假设，将在体内和体外表征三个因素对Pol I活性的贡献。这三个因子在体内与rDNA相关，并且先前已被Pol II涉及转录。本研究将为核糖体生物合成中进化保守转录因子的研究提供新的思路。 Pol I的转录延伸在功能上与rRNA加工偶联。rDNA中的序列元件可以直接影响转录延伸效率和新生rRNA加工。为了检验这一假设，将使用体外单转换延伸率测定和从生长细胞分离的天然延伸转录物的测序来鉴定这些元件。确定的暂停位点的突变和核糖体组装的表征将决定这些序列在rRNA加工中的作用。这一目标将确定DNA元件对所观察到的转录和rRNA加工的编排的贡献。在真核生物中，人们认为转录过程中会发生几个RNA加工事件，该系统可以作为研究这些复杂的共转录过程的模型。 Pol I的转录是抑制癌细胞增殖的有效靶点。为了通过抑制PI I来控制细胞增殖，必须建立对rDNA转录的独特特征的详细理解。这项研究将为正在进行的和未来的项目奠定基础，旨在选择性抑制Pol的癌症化疗。

DESCRIPTION (provided by applicant): Ribosome synthesis is directly linked to the rates of cell growth and proliferation. Transcription of the ribosomal DNA (rDNA) by RNA polymerase I (Pol I) is the first, rate-limiting step in ribosome assembly. In rapidly growing cells, transcription by Pol I accounts for more than 60% of all cellular transcription. The overall hypothesis of this project is that Pol I has evolved sevral unique features that support efficient, robust activity and these features are logical targets for selective inhibition of ribosome synthesis. To test this hypothesis and expand the understanding of ribosome synthesis in eukaryotic cells, this project will focus on three key aspects of rDNA transcription: 1) enzymatic features of Pol I 2) trans-acting factors that control Pol I and 3) sequences in the rDNA that affect transcription elongation and rRNA processing. Pol I has evolved enzymatic properties that suit its cellular role. For example, recent data suggest that the rate-limiting steps in Pol I versus Pol II transcription elongation may differ. These differences could serve as targets for selective inhibition of ribosome biosynthesis. This project will employ rapid mixing kinetic measurements and modeling of resulting data sets to quantitatively describe Pol I transcription elongation. Additional studies will define the elemental steps in transcription elongation that are influenced by specific subunits or domains of the RNA polymerase. Pol I transcription requires trans-acting factors to support the rate of transcription observed in vivo. To test this hypothesis, contributions of three factors to Pol I activity will be characterized in ivo and in vitro. These three factors associate with the rDNA in vivo and have been implicated previously in transcription by Pol II. This study will identify new roles for evolutionary conserve transcription factors in ribosome biogenesis. Transcription elongation by Pol I is functionally coupled to rRNA processing. Sequence elements in the rDNA may directly influence transcription elongation efficiency and nascent rRNA processing. To test this hypothesis, these elements will be identified using single-turnover elongation rate assays in vitro and sequencing of native- elongating transcripts isolated from growing cells. Mutation of identified pause sites and characterization of ribosome assembly will determine the role of these sequences in rRNA processing. This aim will identify the contribution of DNA elements to the observed orchestration of transcription and rRNA processing. In eukaryotes, several RNA processing events are thought to occur during transcription and this system may serve as a model for the study of these complicated co-transcriptional processes. Transcription by Pol I is a validated target for inhibition of cancer cell proliferation. In order to control cell proliferation via inhibition of Pl I, a detailed understanding of the unique features of rDNA transcription must be established. This study will lay the foundation for ongoing and future projects aimed at selective inhibition of Pol for cancer chemotherapy.