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、抗癌药物发现和设计以及衰老机制的理解中具有重要意义。端粒酶是一种非常重要的逆转录酶，它以自身的RNA亚基为模板合成位于真核生物染色体3'端的DNA。端粒DNA是端粒酶活性的产物，是一条高重复、富含鸟嘌呤的单链DNA，是称为端粒的特化核蛋白复合物的一部分。端粒维持是必不可少的，因为端粒保护染色体末端免受DNA修复机制的错误识别和溶核降解。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亚基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循环的机制，这将使端粒酶的结构与广泛的核酸聚合酶以及核糖核蛋白复合物之间进行更准确的比较。具体而言，端粒酶RNA的二级结构将阐明位点特异性突变体和高分辨率的生化技术的组合使用四膜虫嗜热端粒酶RNA（tTR）作为模型。第一个具体目标将使用一种新的单核苷酸分辨率足迹技术，称为选择性引物延伸2 '-羟基酰化（SHAPE），以揭示在任何核苷酸被添加到端粒DNA的3'端之前与端粒酶的逆转录酶结构域复合的tTR的二级结构。第二个目标将使用SHAPE来揭示tTR的二级结构，在tTR被逆转录成端粒DNA重复序列后，与端粒酶的逆转录酶结构域的构象复合。最后的具体目标将测试一套特定的生化技术，以捕获端粒酶的构象相对于核苷酸添加和核苷酸聚合的过程。然后，SHAPE技术将用于揭示这些过程中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.