Control of RNA polymerase I transcription initiation and elongation

描述(由申请人提供)： 核糖体的合成与细胞的生长和增殖速度直接相关。RNA聚合酶I(Pol I)转录核糖体DNA(RDNA)是核糖体组装的第一步，也是限速步骤。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已经进化出几个支持高效、稳健活性的独特功能，这些功能是选择性抑制核糖体合成的合乎逻辑的靶标。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，本项目将重点研究rDNA转录的三个关键方面：1)PolI的酶特性；2)控制PolI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已经进化出与其细胞角色相适应的酶特性。例如，最近的数据表明， POL I和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对所得数据集的建模，以定量描述Pol I转录延伸。更多的研究将确定转录的基本步骤 受RNA聚合酶的特定亚基或结构域影响的伸长。POL I转录需要反式作用因子来支持体内观察到的转录速度。为了验证这一假设，三个因素对POL I活性的贡献将在体内和体外进行表征。这三个因子在体内与rDNA相关，并已被POL II参与转录。本研究将确定进化保守转录因子在核糖体生物发生中的新作用。Pol I的转录延伸在功能上与rRNA加工相连。RDNA中的序列元件可能直接影响转录延伸效率和新生的rRNA加工。为了验证这一假设，将使用体外单周转延伸率分析和从生长细胞中分离的天然延长转录本的测序来鉴定这些元件。确定的停顿位点的突变和核糖体组装的特征将决定这些序列在rRNA加工中的作用。这一目的将确定DNA元件对观察到的转录协调和rRNA加工的贡献。在真核生物中，人们认为在转录过程中会发生几个RNA加工事件，这个系统可以作为研究这些复杂的共转录过程的模型。Pol I转录是抑制癌细胞增殖的有效靶点。为了通过抑制Pl I来控制细胞的增殖，必须对rDNA转录的独特功能有一个详细的了解。这项研究将为正在进行的和未来旨在选择性抑制POL用于癌症化疗的项目奠定基础。

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.