The structure of telomerase RNA during catalysis

描述(由申请人提供)：端粒酶是一种重要的酶，在干细胞技术、抗癌药物的发现和设计以及对衰老机制的理解方面具有重要意义。端粒酶是一种重要的逆转录酶，它以其完整的RNA亚基为模板合成位于真核染色体3‘端的DNA。端粒酶活性的产物端粒DNA是高度重复的富含鸟嘌呤的DNA的单链，是称为端粒的特殊核蛋白复合体的一部分。端粒的维持是必不可少的，因为端粒保护染色体末端不被DNA修复机制错误识别和核溶解降解。端粒酶的几个特征仍然是个谜。这些研究的完成将阐明端粒酶RNA亚单位二级结构的几个特征。拟议的研究还将确定端粒酶催化反应周期的机制，这将使端粒酶与广泛的核酸聚合酶以及核糖核蛋白复合体的结构进行更准确的比较。具体地说，端粒酶RNA的二级结构将以嗜热四膜虫端粒酶RNA(TTR)为模型，通过定点突变和高分辨生化技术相结合来阐明。第一个特定目的是使用一种新的单核苷酸拆分足迹技术，称为选择性2‘-羟基酰化引物延伸(SHAPE)，在端粒DNA的3’端添加任何核苷酸之前，揭示TTR与端粒酶逆转录酶结构域的二级结构。第二个目的是利用SHAPE揭示TTR被逆转录成端粒DNA重复序列后与端粒酶逆转录酶结构域形成的复合体中的二级结构。最终的特定目标将测试一套特定的生化技术，以捕获与核苷酸加成和核苷酸聚合过程相关的构象中的端粒酶。然后，将使用形状技术来揭示在这些过程中TTR的二级结构。

DESCRIPTION (provided by applicant): Telomerase is an important enzyme with implications in stem cell technology, anti cancer drug discovery and design, and understanding of the mechanisms of aging. Telomerase is a remarkable reverse transcriptase that uses its integral RNA subunit as the template for the synthesis of the DNA located at the 3' end of eukaryotic chromosomes. The product of telomerase activity, telomeric DNA, is a single strand of highly repetitive, guanine-rich DNA that is part of the specialized nucleoprotein complex called the telomere. Telomere maintenance is essential because the telomere guards the chromosome ends from mistaken recognition by the DNA repair machinery and nucleolytic degradation. Several features of telomerase remain mysterious. Completion of the studies proposed will illuminate several features of the secondary structure of the RNA subunit of telomerase. The proposed research will also determine the mechanism of the telomerase catalytic reaction cycle, which will allow a more accurate comparison between the structure of telomerase with a broad range of nucleic acid polymerases as well as ribonucleoprotein complexes. Specifically, the secondary structure of telomerase RNA will be elucidated by a combination of site-specific mutants and high resolution biochemical techniques using Tetrahymena thermophila telomerase RNA (tTR) as a model. The first specific aim will use a novel, single nucleotide resolution footprinting technique called selective 2'-hydroxyl acylation by primer extension (SHAPE) to reveal the secondary structure of tTR in complex with the reverse transcriptase domain of telomerase before any nucleotides have been added to the 3' end of telomeric DNA. The second aim will use SHAPE to reveal the secondary structure of tTR in complex with the reverse transcriptase domain of telomerase in conformations after tTR has been reverse transcribed into a telomeric DNA repeat. The final specific aim will test a specific set of biochemical techniques to trap telomerase in conformations relative to the processes of nucleotide addition and nucleotide polymerization. SHAPE technology will then be used to reveal the secondary structure of tTR during these processes.