TELOMERASE RNP STRUCTURE/FUNCTION

真核细胞的染色体末端由端粒、DNA-蛋白质组成 基因组稳定性所必需的结构。实现以下目标的主要机制 合成端粒的是端粒酶，一种不寻常的核糖核蛋白 (RNP)具有逆转录酶活性。在高等真核生物中， 端粒酶的表达受到高度调控，并与 具有无限增殖能力的细胞。例如，超过 90%的人类肿瘤是端粒酶阳性的。尽管这些 观察表明，端粒酶在真核生物中发挥着关键作用， 令人惊讶的是，人们对它的亚基组成知之甚少 酶或其在体内的相互作用。我们正在研究端粒酶 纤毛虫原生动物，Eplotes Crassus。因为独一无二的 基因组的组织，纤毛虫，如游走虫是一种极端的 丰富的端粒来源和为数不多的端粒同时存在的系统之一 很容易观察到维持和从头形成。我们假设 克氏杆菌端粒酶以全酶形式存在，由一种 核心RNP和改变酶行为的因素。我们的假设是 根据初步数据显示1)端粒酶执行从头开始 只在生命周期的一个阶段合成，这个反应是 由反式作用因子介导；和2)端粒酶存在于 高阶络合物表现出不同于 核心RNP粒子。这个项目的长期目标是 阐明端粒酶RNP组分的功能和相互作用。 利用常规层析、亲和纯化和分子 生物学方法，我们建议确定核心蛋白 克氏杆菌端粒酶RNP亚基，克隆相应基因 并产生重组蛋白(目标1)。端粒酶的功能- 相关蛋白质将通过各种生化手段进行检测。 战略，包括开发体外重建系统(AIM 2)。我们将分析更高阶的端粒酶复合体， 相当于端粒酶的全酶，通过提纯这些复合体， 分析它们的成分并检测它们的生化特性 (目标3)。最后，我们将研究其分子基础 端粒酶-DNA相互作用中的发育程控开关。这些 研究包括分析核心RNP的结构变异 从生命周期的两个阶段和表征反式作用 参与新的端粒形成的因素(目标4)。这些研究 将对端粒酶的结构和功能有新的认识 并提供关于端粒酶调节的所需信息。

Eukaryotic chromosome ends are capped by teleomeres, DNA-protein structures essential for genome stability. The primary mechanism for synthesizing teleomeres is telomerase, an unusual ribonucleoprotein (RNP) with reverse transcriptase activity. In higher eukaryotes, telomerase expression is highly regulated and strongly correlated with cells having unlimited proliferation capacity. For example, more than 90 percent of human tumors are telomerase positive. Although these observations imply that telomerase plays a critical role in eukaryotes, surprisingly little is known about the subunit composition of this enzyme or its interactions in vivo. We are studying telomerase in a ciliated protozoan, Euplotes crassus. Because of the unique organization of the genome, ciliates such as Euplotes are an extremely rich source of telomeres and one of the few systems where both telomere maintenance and de novo formation are readily observed. We hypothesize that the E. crassus telomerase exists as a holoenzyme, comprised of a core RNP and factors that modify enzyme behavior. Our hypothesis is based on preliminary data showing 1) that telomerase performs de novo synthesis in only one stage of the life-cycle and this reaction is mediated by a trans-acting factor; and 2) that telomerase exists in higher order complexes exhibiting biochemical properties distinct from the core RNP particle. The long-term goal of this project is to elucidate the function and interactions of telomerase RNP constituents. Using conventional chromatography, affinity purification and molecular biological approaches, we propose to identify the core protein subunits of the E. crassus telomerase RNP, clone the corresponding genes and generate recombinant proteins (Aim 1). The function of telomerase- associated proteins will be examined using a variety of biochemical strategies, including development of an vitro reconstitution system (Aim 2). We will analyze higher order telomerase complexes that may correspond to a telomerase holoenzyme, by purifying these complexes, analyzing their constituents and examining their biochemical properties (Aim 3). Finally, we will investigate the molecular basis for the developmentally programmed switch in telomerase-DNA interactions. These studies include assaying for structural variations in the core RNP from two stages of the life cycle and characterizing the trans-acting factor implicated in de novo telomere formation (Aim 4). These studies will generate new insight into the structure and function of telomerase and provide needed information regarding telomerase regulation.