ANALYSIS OF TELOMERASE REVERSE TRANSCRIPTASE

描述（改编自申请人的摘要）：端粒酶是一种 核糖核蛋白复合物，负责延伸富含dG的链， 端粒末端重复序列。它使用一小部分稳定相关的 RNA组分作为模板，预先存在的染色体末端作为引物， 完成逆转录。端粒酶活性被特异性激活 在癌细胞中，并通过赋予细胞 拥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，证实了 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 从分子水平了解端粒酶的结构、机制、组装和 调控这项提议集中在端粒酶复合体的一个关键成分上， 逆转录酶多肽（称为TERT）。位于 这种蛋白质的C-末端一半是必需的逆转录酶样基序 参与聚合反应。其他人的比较序列分析， 首席研究员发现，在蛋白质的N-末端的一半， 新的保守基序（命名为GQ，CP，QFP和T，主要基于关键 每个基序内的不变残基），这些残基可能对其 功能首席研究员假设这些图案介导了 TERT的关键活性，如RNA结合，蛋白质稳定性，复合物 组装、与调节因子的相互作用和逆转录。的 该提案的目标是阐明精确的分子 四个主题的功能。 方法学包括端粒酶复合体的生化和遗传分析 携带缺失和置换突变，以及生物物理和 关键蛋白质结构域的结构表征。鉴于 丰富的生物化学和遗传工具，芽殖酵母 酿酒酵母代表了一个理想的模式系统， 这样的奋进。此外，由于图案的保守性， 通过分析，这些发现应该可以为端粒酶提供一般性的见解。 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.