ANALYSIS OF TELOMERASE REVERSE TRANSCRIPTASE

描述(摘自申请人摘要)：端粒酶是 核糖核蛋白复合体负责延伸富含DG的链 端粒末端重复。它使用了一小部分稳定关联的 以RNA组分为模板，以已有的染色体末端为引物 完成逆转录。端粒酶活性被特别激活 在癌细胞中，并通过赋予细胞促进肿瘤转化 具有无限的复制潜力。端粒酶的特异性失活 肿瘤细胞导致端粒缩短和凋亡，验证了 端粒酶抑制剂在抗癌治疗中的潜力。 这项研究计划的长期目标是建立一个详细的 端粒酶结构、机制、组装和端粒酶的分子理解 监管。这项建议侧重于端粒酶复合体的一个关键成分， 逆转录酶多肽(简称TERT)。位于 该蛋白的C末端的一半是必需的逆转录酶样基序 参与聚合的物质。其他人的比较序列分析和 首席研究员揭示了在N端的蛋白质4的一半 新的保守基序(命名为GQ、CP、QFP和T，主要基于key 每个基序中的不变残基)，这些残基可能对其 功能。首席调查者假设这些主题在 TERT的关键活性，如RNA结合、蛋白质稳定性、复合体 组装，与调控因子的相互作用，以及逆转录。这个 这一提议的目的是为了阐明精确的分子 所有四个主题的功能。 该方法包括对端粒酶复合体的生化和遗传分析。 携带缺失和替换突变，以及生物物理和 分离中关键蛋白质结构域的结构特征。给定 丰富的可用生化和遗传工具，萌芽酵母 酿酒酵母是一种理想的创业模式系统 这样的努力。此外，由于主题的保守性， 通过分析，这些发现应该会提供关于端粒酶的总体见解。 结构和机制。

DESCRIPTION (adapted from applicant's abstract): Telomerase is a ribonucleoprotein complex responsible for extending the dG-rich strand of the telomere terminal repeats. It employs a small segment of a stably associated RNA component as template, and pre-existing chromosomal ends as primers to accomplish reverse transcription. Telomerase activity is specifically activated in cancer cells, and promotes neoplastic transformation by conferring cells with unlimited replicative potentials. Specific inactivation of telomerase in tumor cells leads to telomere shortening and apoptosis, validating the potential of telomerase inhibitors in anti-cancer therapy. The long-term objective of this research plan is to establish a detailed molecular understanding of telomerase structure, mechanism, assembly and regulation. This proposal focuses on a key component of the telomerase complex, the reverse transcriptase polypeptide (known as TERT). Located within the C-terminal half of this protein are essential reverse-transcriptase-like motifs that participate in polymerization. Comparative sequence analysis by others and the principal investigator revealed in the N-terminal half of the protein four novel conserved motifs (named GQ, CP, QFP, and T based primarily on key invariant residues within each motif) that are likely to be critical to its function. The principal investigator hypothesizes that these motifs mediate crucial activities of TERT, such as RNA-binding, protein stability, complex assembly, interaction with regulatory factors, and reverse transcription. The goals of this proposal are directed toward elucidating the precise molecular functions of all four motifs. The methodology involves biochemical and genetic analysis of telomerase complex bearing deletion and substitution mutations, as well as biophysical and structural characterization of critical protein domains in isolation. Given the wealth of available biochemical and genetic tools, the budding yeast Saccharomyces cerevisiae represents an ideal model system for undertaking such an endeavor. Furthermore, because of the conserved nature of the motifs being analyzed, the findings should provide general insights on telomerase structure and mechanisms.