The Structure of Telomerase RNA Attending Catalysis

通过补充每条染色体3'端的DNA，端粒酶是细胞不朽的关键之一。在功能上，端粒酶是一种重要的核酸聚合酶，它以其完整的RNA亚基为模板，合成位于真核生物染色体3'端的DNA。端粒酶的产物，端粒DNA，是一条高度重复的富含g的DNA单链，是称为端粒的特殊核蛋白复合体的一部分。端粒维护是必不可少的，因为端粒保护染色体末端免受DNA修复机制的错误识别和随后的核分解降解。端粒酶催化机制的几个特点仍然是神秘的，本项目的研究应阐明端粒酶功能的结构基础。具体而言，以嗜热四膜虫端粒酶RNA （tTR）为模型，研究者将通过使用各种高分辨率生化技术阐明端粒酶RNA的作用。在第一个具体目标中，tTR的结构将在溶液和催化活性配合物中进行研究，使用引物延伸（SHAPE）分析选择性2'-羟基酰化。在第二个目标中，整个tTR中的重要官能团将使用核苷酸模拟干扰作图来确定，这是一种高通量方法，允许同时询问RNA的每个核苷酸的官能团。在最终目标中，根据目标1和目标2的数据设计的tTR位点特异性突变体将用于证实这些发现。这项研究将由学生进行，包括科学界代表性不足的群体的成员。此外，参加少数民族暑期研究项目的学生也应参与本项目。该项目的资金将为学生提供用品和研究津贴。此外，端粒酶是影响包括衰老在内的许多生物现象的重要酶，因此对它的了解可以在许多方面造福社会。

By replenishing the DNA at the 3' end of each chromosome, the enzyme telomerase is one key to cellular immortality. Functionally, telomerase is a remarkable nucleic acid polymerase 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, telomeric DNA, is a single strand of highly repetitive G-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 consequent nucleolytic degradation. Several features of the catalytic mechanism of telomerase remain mysterious, and the research conducted in this project should illuminate the structural basis for telomerase function. Specifically, using Tetrahymena thermophila telomerase RNA (tTR) as a model, the investigator will elucidate the role of telomerase RNA through the use of a variety of high resolution biochemical techniques. In the first specific aim the structure of tTR will be studied both in solution and in the catalytically active complex using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In the second aim important functional groups throughout tTR will be identified using nucleotide analog interference mapping, a high throughput approach that allows simultaneous interrogation of functional groups of each nucleotide of an RNA. In the final aim, site-specific mutants of tTR, designed based on data from aims 1 and 2, will be used to corroborate those findings.This research will be conducted by students, including members of groups underrepresented in science. Furthermore, students who participate in a minority summer research program are expected to also participate in this project. Funding for the project will provide students with supplies and research stipends. Furthermore, telomerase is an important enzyme that affects many biological phenomena, including that of aging, and thus its understanding can benefit society in many ways.