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Investigating Telomerase Mechanism by Exploring the RNA

通过探索 RNA 研究端粒酶机制

基本信息

批准号：
7878503
负责人：
David Clifford Zappulla
金额：
$ 24.65万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-15 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7878503
关键词：
Aging Back Binding Binding Sites Biochemistry Biological Assay Cell Cycle Cells Chromosomal Breaks Chromosomes Collaborations Colorado Communities Complex Consensus Coupled DNA DNA repair protein Development Plans Disadvantaged Disease Doctor of Philosophy Drug Delivery Systems Educational process of instructing Enzymes Excision Faculty Goals Human In Vitro Individual Knowledge Learning Length Malignant Neoplasms Modeling Multienzyme Complexes Nucleotides Pathway interactions Phase Phenotype Play Positioning Attribute Process Protein Subunits Proteins RNA RNA-Directed DNA Polymerase Reaction Recruitment Activity Regulation Relative (related person)Research Ribonucleoproteins Role Rolfing Saccharomyces cerevisiae Structure System T-Lymphocyte Telomerase Telomerase RNA Component Temperature Testing Training Universities Work Yeasts cancer cell career development design enzyme mechanism fitness flexibility helicase human disease in vitro activity in vivo inhibitor/antagonist insight miniaturize neoplastic cell nuclease protein function reconstitution research study scaffold skills telomere tool

项目摘要

After studying transcriptional silencing and DMA replication during my PhD 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 nonprocessive 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.

在我与罗尔夫的博士论文研究期间，我研究了转录沉默和DMA复制在石溪大学，我搬到了科罗拉多大学汤姆·切赫的实验室博士后工作。我的目标是加深我的生物化学知识，特别是学习更多关于RNA，利用切赫实验室和CU-Boulder的互动RNA社区的专业知识。我的研究涉及到端粒酶，它复制真核染色体的末端，由一个 RNA以及几个蛋白质亚基。端粒酶在人类疾病中发挥着重要作用，如癌症。在我的博士后工作中，我已经开发了1157-NT酵母的二级结构模型端粒酶RNA、TLC1，并首次进行了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体中RNA支架的灵活性，并开始明确重要的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，部分是通过确定Ku是否灵活通过移动其结合位点，然后测试功能，以RNA为支架。我也会测试绝对的 TLC1 RNA大小的限制和对端粒酶RNA二级结构的建议共识。我的职业发展计划包括学习RNA结构是如何通过计算手段预测的，将为这项研究提供便利。具体目标#2部分描述了检验酵母端粒酶核心酶在体外是非过程性的，因为反复招募和移除酶的因子体内没有底物。已经与其他实验室合作，以获得必要的纯化的蛋白质在化验中进行测试。最后，在具体目标#3中，我计划研究和开发表达小型化端粒酶RNA Mini-T的细胞，并建议鉴定相关的抑制子我已经确认了这一点。通过检验这些假设和学习新的研究技能课程作业和小长假，以及获得教学学位和其他相关培训，我希望达到我的目标，准备好随后担任教员职位。相关性：端粒酶是一种必须仔细调节才能正常运行的酶，否则就会可能在导致癌症等疾病方面发挥关键作用。因为端粒酶的异常过度生产被认为是~90%的人类癌细胞无限增殖的关键，端粒酶是一种有吸引力的药物靶点，尽管很难找到合适的抑制剂，因此获得更多信息关于端粒酶的功能是必需的。在这里，我描述了推导出以下最低要求的实验酵母端粒酶功能，一个遗传和生化上有利的系统，有望继续阐明人类酶的特征。