Investigating Telomerase Mechanism by Exploring the RNA

描述（由申请人提供）：我在石溪大学跟随Rolf Sternglanz博士论文研究转录沉默和DNA复制后，转到科罗拉多大学Tom切赫的实验室进行博士后工作。我的目标是加深我的生物化学知识，特别是更多地了解RNA，利用切赫实验室和科罗拉多大学博尔德分校的互动RNA社区的专业知识。我的研究涉及端粒酶，它复制真核生物染色体的末端，由RNA和几个蛋白质亚基组成。端粒酶在癌症等人类疾病中起着重要作用。在我的博士后工作中，我已经开发了1157-nt酵母端粒酶RNA TLC1的二级结构模型，并首次在体外重建酵母端粒酶活性测定。我现在建议使用这些工具来检查酵母端粒酶复合体中RNA支架的灵活性，并开始定义重要辅助蛋白在酶机制中的作用。在具体目标1中，我计划研究酵母端粒酶RNA功能的结构要求，部分方法是通过移动其结合位点来确定Ku是否被RNA灵活地支架化，然后测试功能。我还将测试TLC1 RNA大小的绝对局限性和端粒酶RNA二级结构的拟议共识。我的职业发展计划包括学习如何通过计算手段预测RNA结构，这将有助于这项研究。具体目标2部分描述了测试酵母端粒酶核心酶在体外是非进程性的假设，因为在体内缺乏从底物中迭代地招募和去除酶的因素。已与其他实验室合作，以获得必要的纯化蛋白质，以便在分析中进行测试。最后，在具体目标3中，我计划检查和利用表达小型化端粒酶RNA （Mini-T）的细胞的表型，并提出表征我已经确定的相关抑制因子。通过测试这些假设，并通过课程作业和短期休假学习新的研究技能，以及获得一定程度的教学和其他相关培训，我希望实现我的目标，为随后担任教师职位做好准备。

DESCRIPTION (provided by applicant): After studying transcriptional silencing and DNA replication during my Ph.D. thesis research with Rolf Sternglanz at Stony Brook University, I moved to Tom Cech's lab at the University of Colorado for postdoctoral work. My goal has been to deepen my knowledge of biochemistry and specifically to learn more about RNA, drawing on the expertise of the Cech lab and the interactive RNA community at CU-Boulder. My research concerns telomerase, which replicates the ends of eukaryotic chromosomes and is composed of an RNA as well as several protein subunits. Telomerase plays a prominent role in human diseases such as cancer. Already in my postdoctoral work I have developed a secondary structure model for the 1157-nt yeast telomerase RNA, TLC1, and the first in vitro reconstituted yeast telomerase activity assay. I now propose to use these tools to examine the flexibility of RNA scaffolding in the yeast telomerase complex and to begin to define the roles of important accessory proteins in the enzyme mechanism. In specific aim 1, I plan to investigate structural requirements for yeast telomerase RNA function, in part by determining if Ku is flexibly scaffolded by the RNA by moving its binding site and then testing functionality. I will also test the absolute limitations of TLC1 RNA size and the proposed consensus for telomerase RNA secondary structure. My career development plan involves learning how RNA structure is predicted by computational means, which will facilitate this research. Specific aim 2 describes, in part, testing the hypothesis that yeast telomerase core enzyme is non-processive in vitro because factors that iteratively recruit and remove the enzyme from its substrate in vivo are absent. Collaboration with other labs has been undertaken to acquire the necessary purified proteins to test in the assay. Lastly, in specific aim 3, I plan to examine and exploit phenotypes of cells expressing the miniaturized telomerase RNA, Mini-T, and propose to characterize a related suppressor that I have already identified. By testing these hypotheses and learning new research skills through coursework and a mini-sabbatical, as well as gaining a degree of teaching and other relevant training, I expect to attain my goal of becoming prepared to subsequently assume a faculty position. Relevance: Telomerase is an enzyme that must be carefully regulated to perform appropriately, or else it can play a pivotal role in causing diseases such as cancer. Because abnormal overproduction of telomerase is thought to be critical for unlimited proliferation of ~90% of human cancer cells, telomerase is an attractive drug target, although it has been very difficult to find an appropriate inhibitor and therefore more information about telomerase function is needed. Here I describe experiments to deduce the minimal requirements of yeast telomerase function, a genetically and biochemically advantageous system that is expected to continue to illuminate features of the human enzyme.