TELOMERASE RIBONUCLEOPROTEIN STRUCTURE

端粒是一种特殊的DNA-蛋白质结构，覆盖在端粒的末端 真核细胞的染色体，并在维持 染色体的稳定性和适当的核结构。高度保守 在整个进化过程中，端粒DNA由简单的重复序列组成 序列。一种名为端粒酶的新型核糖核蛋白(RNP)酶 负责合成端粒DNA。在端粒酶RNA中 部分是与端粒重复序列互补的序列，这 结构域是DNA合成的模板。因此，端粒酶是一种 专门化类型的逆转录酶。这样做的长期目标是 该项目旨在阐明端粒合成的机制，通过 端粒酶。目前，我们对这一过程的理解是 受阻于缺乏关于 端粒酶RNP。虽然RNA和蛋白质都是活性所必需的， 端粒酶RNA只有部分特征，而 该酶的蛋白质组分(S)尚未鉴定。这 应用的目的是进一步分析端粒酶RNP的结构 利用我们手中的组件，RNA部分。这个 为这些研究选择的实验系统是下毛纤毛虫， Eplotes和Oxytricha是已知的两个最丰富的端粒来源。 这项提议有两个主要目标。第一个是考试。 分析端粒酶RNA的二级结构 通过系统发育和体外实验的方法。此外，我们 还将探索端粒模板结构域在 Oxytricha端粒酶RNA。这份提案的第二部分调查了 端粒酶RNP中的RNA-蛋白质相互作用。两种不同的 基于开发的技术来分析其他小的 核RNP复合体将被用来识别轻微 与端粒酶RNA相关。第一个将是部分 用常规柱层析法纯化游离体端粒酶RNP 层析和平衡梯度离心法 酶活性或端粒酶RNA成分作为标记物 端粒酶复合体。样品将通过亲和进一步提纯 利用寡核苷酸筛选端粒酶RNP颗粒 与端粒酶RNA部分互补。第二个战略是 鉴定与端粒酶RNA检测相关的多肽 从部分纯化的端粒酶制剂中组装蛋白质 外源端粒酶RNA转录本的体外实验。结合蛋白质 将对端粒酶RNA进行鉴定和进一步鉴定。

Telomeres are the specialized DNA-protein structures that cap the ends of eukaryotic chromosomes and serve an essential role in maintaining chromosome stability and proper nuclear architecture. Highly conserved throughout evolution, telomeric DNA consists of simple tandemly repeated sequences. A novel ribonucleoprotein (RNP) enzyme called telomerase is responsible for synthesizing telomeric DNA. Within the telomerase RNA moiety is a sequence complementary to the telomeric repeats and this domain serves as the template for DNA synthesis. Thus, telomerase is a specialized type of reverse transcriptase. The long range goal of this project is to elucidate the mechanism of telomere synthesis by the telomerase enzyme. Currently, our understanding of this process is impeded by the lack of information concerning the components of the telomerase RNP. Although both RNA and protein are required for activity, the telomerase RNA has only been partially characterized, while the protein component(s) of this enzyme have not yet been identified. This application is aimed at further analysis of telomerase RNP structure by exploiting the component we have in hand, the RNA moiety. The experimental system chosen for these studies are hypotrichous ciliates, Euplotes and Oxytricha, two of the richest known sources of telomeres. There are two major goals in this proposal. The first is an examination of the telomerase RNA, analyzing the secondary structure of this molecule by phylogenetic and in vitro experimental approaches. In addition, we will also explore the function of the telomeric templating domain in the Oxytricha telomerase RNA. The second part of this proposal investigates RNA-protein interactions within the telomerase RNP. Two different approaches based on techniques developed for the analysis of other small nuclear RNP complexes will be used to identify proteins that are lightly associated with the telomerase RNA. The first will be to partially purify the Euplotes telomerase RNP using conventional column chromatography and equilibrium gradient centrifugation, following either enzymatic activity or the telomerase RNA component as a marker for the telomerase complex. Samples will be further purified by affinity selection of Euplotes telomerase RNP particles using oligonucleotides complementary to the telomerase RNA moiety. A second strategy to identify polypeptides associated with the telomerase RNA will assay for the assembly of proteins from partially purified telomerase preparations onto exogenous telomerase RNA transcripts in vitro. Proteins that bind to the telomerase RNA will be identified and further characterized.