NMR Studies of Tetrahymena Telomerase RNA

端粒酶是一种大的、多亚单位的核糖核蛋白复合物，负责染色体物理末端的端粒DNA的复制。端粒酶是由RNA成分，包括DNA合成的模板，催化蛋白质成分，和各种其他蛋白质。由于端粒酶在大多数生物体中丰度低，并且难以分离完整的全酶，因此其确切的蛋白质组成尚不清楚，并且所鉴定的蛋白质和RNA组分在RNP加工、组装和功能中的作用仅被部分表征。与脊椎动物和酵母端粒酶的非常大的RNA组分相比，纤毛四膜虫端粒酶的长度为159 nt。嗜热四膜虫的转录活性大核具有约40，000个端粒，因此端粒酶比其他生物更丰富。这有助于四膜虫端粒酶全酶的生化表征。由于四膜虫RNA的体积较小，以及最近在分离全酶组分方面的进展，四膜虫为端粒酶的结构研究提供了一个很好的模型系统。该项目将集中在整个T的结构表征。嗜热菌端粒酶及其与端粒酶的一些蛋白质组分的相互作用，主要使用NMR光谱。虽然端粒酶逆转录酶催化核苷酸添加，但RNA并不简单地发挥支架的作用，而是端粒酶核苷酸和重复添加持续合成能力所必需的。具体目标是：（1）确定茎环IV的结构并表征其与端粒酶逆转录酶的RNA结合结构域、端粒酶组装蛋白p65和假结的相互作用：（2）确定四膜虫和酵母的假定假结的结构并将其结构和功能与人端粒酶假结进行比较;（3）研究含有模板和模板邻近区域的大的未表征的“单链”环的结构及其与茎环IV的相互作用。这些结构域研究的信息将用于帮助确定全长端粒酶的结构。该项目将为本科生、研究生和博士后研究员提供核磁共振结构生物学和核酸及核酸-蛋白质复合物生物物理学方面的培训。这是一个妇女和少数民族长期以来代表性不足的领域，PI的实验室为他们提供了一个榜样。这些研究的结果可能最终会出现在生物化学教科书中; Voet和Voet Biochemistry（第3版）包括PI的2种结构。

Telomerase is a large, multi-subunit ribonucleoprotein complex that is responsible for replication of telomere DNA found at the physical ends of chromosomes. Telomerase is composed of an RNA component that includes the template for DNA synthesis, a catalytic protein component, and a variety of other proteins. Due to the low abundance of telomerase in most organisms and the difficulties in isolating the intact holoenzyme, its exact protein composition is not yet known, and the roles of identified protein and RNA components in RNP processing, assembly, and function have only been partially characterized. In contrast to the very large RNA component of vertebrate and yeast telomerases, the ciliate Tetrahymena telomerase is 159 nts long. The transcriptionally active macronucleus of Tetrahymena thermophila has ~40,000 telomeres, and thus telomerase is more abundant than in other organisms. This has facilitated biochemical characterization of the Tetrahymena telomerase holoenzyme. Because of the smaller size of its RNA and the recent progress in isolating the components of the holoenzyme, Tetrahymena provides a good model system for structural studies of telomerase. This project will focus on structural characterization of the entire T. thermophila telomerase and its interactions with some of the protein components of telomerase using primarily NMR spectroscopy. Although telomerase reverse transcriptase catalyzes nucleotide addition, the RNA does not simply play a role as a scaffold, but rather is essential for telomerase nucleotide and repeat addition processivity. The specific objectives are: (1) Determine the structure of stem-loop IV and characterize its interactions with the RNA binding domain of telomerase reverse transcriptase, the telomerase assembly protein p65, and the pseudoknot; (2) Determine the structures of the putative pseudoknots of Tetrahymena and yeast and compare their structure and function to the human telomerase pseudoknot; (3) Investigate the structure of the large uncharacterized 'single stranded' circle that contains the template and template adjacent regions and its interactions with stem-loop IV. The information from these domain studies will be used to help determine the structure of the full length telomerase. This project will provide training to undergraduate and graduate students and postdoctoral fellows in NMR structural biology and biophysics of nucleic acids and nucleic acid-protein complexes. This is an area in which women and minorities have long been underrepresented, and the PI's lab provides a role model for them. Results from these studies will likely end up in Biochemistry textbooks; Voet and Voet Biochemistry(3rd edition) includes 2 of the PI's structures.