Structure and dynamics of the Tetrahymena telomerase ribonucleoprotein

描述（由申请人提供）：端粒酶核糖核蛋白（RNP）是维持端粒所必需的，端粒是保护真核细胞染色体末端免受异常加工和有害的端对端融合事件影响的特化核蛋白结构。端粒酶催化端粒DNA的进行性延伸，使用一种特殊的催化机制，需要端粒酶RNA，端粒酶逆转录酶（TERT）和几个额外的蛋白质亚基的强功能相互依赖性。本研究的主要目的是阐明端粒酶RNA和蛋白质亚基中保守结构域如何协调端粒酶RNP组装和催化过程。为了解决与端粒酶的结构分析相关的重大挑战，我们将研究端粒酶复合物从成熟的模式生物嗜热四膜虫，使用多方面的实验策略，结合单分子生物物理技术与计算，生物化学和高分辨率的结构方法配对。在目标1中，我们将使用化学RNA探针和单分子Forster共振能量转移（smFRET）来表征端粒酶RNA溶液的结构和动力学，分别。从这些实验中出现的距离约束将用于指导与Nikolai Ulyanov（UCSF）合作的RNA结构预测计算。在目标2中，我们将使用靶向羟基自由基探测、基于smFRET的结构测量和X射线晶体学来确定核心端粒酶RNP内的保守RNA和蛋白质结构域的三维组织。这项工作将与Kathleen柯林斯（UCB）和Harry Noller（UCSC）合作进行。在目标3中，我们将开发一种新的单分子端粒酶结构-功能分析，以严格评估现有的模型端粒酶构象动力学过程中端粒DNA合成。在大多数细胞中，端粒长度随着每一轮细胞分裂而逐渐缩短，为细胞衰老提供了分子信号，并调节进入永久性细胞生长停滞。相反，具有高水平增殖能力的细胞（即干细胞）通过端粒酶的酶促作用维持端粒长度。了解端粒酶的分子机制和调控具有直接的医学意义，因为端粒酶功能障碍有助于人类疾病，包括早衰综合征和大多数癌症。因此，端粒酶研究的目的是开发新的方法来诊断和治疗端粒酶相关疾病。 公共卫生相关性：端粒酶是维持端粒所必需的酶，端粒是在染色体末端发现的保护性帽状结构。端粒酶缺陷出现在大多数（~90%）人类癌症和几种早衰综合征中。因此，努力更好地理解端粒酶的机制和调节的动机是开发用于诊断和治疗端粒酶相关疾病的新方法的目标。为此，该建议旨在阐明端粒酶的结构特性，这些特性是其独特的细胞活性的基础。

DESCRIPTION (provided by applicant): The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fusion events. Telomerase catalyzes the processive extension of telomere DNA using a specialized catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase (TERT), and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural domains within telomerase RNA and protein subunits coordinate the processes of telomerase RNP assembly and catalysis. To address the substantial challenges associated with structural analysis of telomerase we will study the telomerase complex from the well-established model organism Tetrahymena thermophila, using a multifaceted experimental strategy that combines single molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. In aim 1, we will use chemical RNA probing and single molecule Forster resonance energy transfer (smFRET) to characterize the telomerase RNA solution structure and dynamics, respectively. Distance constraints that emerge from these experiments will be used to guide RNA structure prediction calculations in collaboration with Nikolai Ulyanov (UCSF). In aim 2, we will determine the three dimensional organization of conserved RNA and protein domains within the core telomerase RNP using targeted-hydroxyl radical probing, smFRET-based structure measurements, and x-ray crystallography. This work will be conducted in collaboration with Kathleen Collins (UCB) and Harry Noller (UCSC). In aim 3, we will exploit a novel single molecule telomerase structure-function assay to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis. In most cells, a progressive shortening of telomere length with each round of cell division provides a molecular signal for cell aging and regulates entry into permanent cell growth arrest. In contrast, cells possessing a high level of proliferative capacity (i.e. stem cells) maintain telomere length through the enzymatic action of telomerase. Understanding the molecular mechanism and regulation of telomerase is of direct medical significance because telomerase dysfunction contributes to human disease, including premature aging syndromes and the majority of cancers. Thus, telomerase research is motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. PUBLIC HEALTH RELEVANCE: Telomerase is an essential enzyme required for maintaining telomeres, the protective capping structures found at chromosome ends. Telomerase defects arise in the majority (~90%) of human cancers and several premature aging syndromes. Thus, efforts to better understand the mechanism and regulation of telomerase are motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. To this end, this proposal aims to illuminate the structural properties of telomerase that underlie its unique cellular activity.