Structural Studies of Telomerase Holoenzyme

描述（由申请人提供）：端粒酶是负责维持端粒的核糖核蛋白复合物，端粒是线性染色体末端的长串串联重复DNA和相关结合蛋白。端粒保护染色体的末端不被细胞DNA修复机制识别为双链断裂，并且还防止端对端连接。在没有活性端粒酶的情况下，端粒随着每一轮DNA复制而缩短。一旦端粒的临界量丢失，细胞进入复制性衰老，有时会发生凋亡。大多数体细胞中的端粒酶表达是检测不到的，而大多数癌细胞恰恰相反，允许它们无限复制。这种活性差异使得端粒酶成为抗增殖药物的一个有吸引力的靶点。该奖学金的目标将是获得四膜虫嗜热端粒酶全酶复合物的冷冻电子显微镜结构，分配每个组件，从催化循环中不同点获得的结构推断功能，并通过NMR和X射线晶体学解决p19亚基的结构。在复制过程中，T.嗜热菌的染色体片段分为约20，000个亚染色体，每个亚染色体都有自己的端粒。为了维持如此多的端粒，T.嗜热菌也表达大量的端粒酶。这确保了它作为模式生物的用途。具体目的是：（1）纯化T.（2）通过抗体和亲和标记定位单个蛋白质和RNA亚基，（3）优化冷冻电镜的样品条件并确定冷冻电镜结构，（4）用结合的DNA引物确定四膜虫端粒酶全酶的结构并与未结合的结构进行比较，（5）用核磁共振和晶体学方法确定T.嗜热菌蛋白亚基p19。这些实验将揭示端粒酶全酶的完整结构，并深入了解分子间接触和亚基运动在端粒催化中发挥的作用。

DESCRIPTION (provided by applicant): Telomerase is the ribonucleoprotein complex responsible for maintaining telomeres, the long stretches of tandem repeating DNA and associated binding proteins at the ends of linear chromosomes. Telomeres protect the ends of chromosomes from being recognized by the cellular DNA repair machinery as double strand breaks and also prevent end-to-end joining. In the absence of active telomerase, telomeres shorten with each round of DNA replication. Once a critical amount of the telomere is lost, the cell enters replicative senescence and sometimes undergoes apoptosis. Telomerase expression in most somatic cells is undetectable, with the opposite true of most cancer cells-allowing them to replicate indefinitely. This difference in activity makes telomerase an attractive target for ani-proliferative drugs. The goal of this fellowship will be to acquire a cryoelectron microscopy structure of the Tetrahymena thermophila telomerase holoenzyme complex, assign each component, deduce function from structures obtained at different points in the catalytic cycle, and solve the structure of the p19 subunit by NMR and X-ray crystallography. During replication, the chromosomes of T. thermophila fragment into ~20,000 subchromosomes, each with their own telomeres. To maintain so many telomeres, T. thermophila also expresses a large amount of telomerase. This has ensured its use as a model organism. The specific aims are to (1) purify T. thermophila telomerase holoenzyme from TAP-tagged strains for electron microscopy and obtain a negative stain structure, (2) localize individual protein and RNA subunits by antibody and affinity labeling, (3) optimize sample conditions for cryoelectron microscopy and determine a cryoelectron microscopy structure, (4) determine the structure of Tetrahymena telomerase holoenzyme with bound DNA primer and compare to the unbound structure, and (5) Use NMR and crystallography to determine the structure of the T. thermophila protein subunit p19. These experiments will reveal the complete structure of a telomerase holoenzyme as well as offer insight into what roles intermolecular contacts and subunit movement play in telomere catalysis.