Investigating Telomerase Mechanism by Exploring the RNA

描述（由申请人提供）：在我博士期间研究转录沉默和DNA复制后。在斯托尼布鲁克大学与罗尔夫·斯特恩格兰茨一起进行论文研究之后，我搬到了科罗拉多大学汤姆·切赫的实验室从事博士后工作。我的目标是加深我的生物化学知识，特别是了解更多关于RNA的知识，利用Cech实验室和CU-Boulder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。端粒酶在人类疾病如癌症中起着重要作用。在我的博士后工作中，我已经开发了一个1157-nt的酵母端粒酶RNA，TLC 1的二级结构模型，和第一个体外重组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物中RNA支架的灵活性，并开始确定酶机制中重要辅助蛋白的作用。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，部分通过确定Ku是否通过移动其结合位点由RNA灵活地支撑，然后测试功能。我还将测试TLC 1 RNA大小的绝对限制和端粒酶RNA二级结构的建议共识。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部分地描述了测试酵母端粒酶核心酶在体外是非进行性的假设，因为不存在在体内迭代地从其底物中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进行检测。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。通过测试这些假设和学习新的研究技能，通过课程和一个迷你休假，以及获得一定程度的教学和其他相关培训，我希望达到我的目标，成为准备随后承担教师的位置。 相关性：端粒酶是一种必须仔细调节才能适当发挥作用的酶，否则它可能在引起癌症等疾病方面发挥关键作用。由于端粒酶的异常过度产生被认为是~90%的人类癌细胞的无限增殖的关键，端粒酶是一个有吸引力的药物靶点，尽管很难找到合适的抑制剂，因此需要更多关于端粒酶功能的信息。在这里，我描述的实验来推断酵母端粒酶功能的最低要求，一个遗传和生物化学上有利的系统，预计将继续照亮人类酶的功能。

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.