TELOMERASE RNP STRUCTURE/FUNCTION

真核染色体末端被端粒、DNA-蛋白质 对基因组稳定性至关重要。 的主要机制 端粒是端粒酶，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， 端粒酶的表达是高度调节的， 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。 虽然这些 观察暗示端粒酶在真核生物中起关键作用， 令人惊讶的是，对这种亚基的组成知之甚少。 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，粗壮游仆虫。 因为独特的 在基因组的组织中，纤毛虫如游仆虫是一种极其重要的生物。 端粒的丰富来源，也是少数几个端粒 容易观察到维持和从头形成。 我们假设 该E.厚层端粒酶作为全酶存在，由 核心RNP和改变酶行为的因素。 我们的假设是 基于初步数据显示1）端粒酶从头开始 合成仅在生命周期的一个阶段，该反应是 由反式作用因子介导;和2）端粒酶存在于 更高级的复合物，其生物化学性质不同于 核心RNP粒子 该项目的长期目标是 阐明端粒酶RNP组分的功能和相互作用。 使用常规色谱法、亲和纯化和分子生物学方法， 生物学方法，我们建议确定核心蛋白 E.粗肌端粒酶RNP，克隆相应基因 并产生重组蛋白（目的1）。 端粒酶的功能- 相关的蛋白质将使用各种生物化学方法进行检测。 战略，包括体外重建系统的发展（目的 2）。我们将分析更高级的端粒酶复合物， 对应于端粒酶全酶，通过纯化这些复合物， 分析其成分并检测其生化特性 (Aim 3）。 最后，我们将研究的分子基础， 端粒-DNA相互作用中的发育程序化开关。 这些 这些研究包括对核反应堆核反应堆的结构变化进行分析 从生命周期的两个阶段， 端粒从头形成的相关因子（Aim 4）。 这些研究 将对端粒酶的结构和功能产生新的认识 并提供有关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.