Investigating Telomerase Mechanism by Exploring the RNA

通过探索 RNA 研究端粒酶机制

• 批准号: 7244712
• 负责人: David Clifford Zappulla
• 金额: $ 8.46万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244712
• 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 biosynthesis; 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; Pliability; 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; Thinking; Training; Universities; Work; Yeasts; cancer cell; career; design; enzyme mechanism; fitness; helicase; human disease; in vivo; inhibitor/antagonist; insight; miniaturize; neoplastic cell; nuclease; protein function; reconstitution; research study; scaffold; size; skills; telomere; tool

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.

描述(申请人提供)：在石溪大学与罗尔夫·斯特恩格朗兹一起研究博士论文期间，我研究了转录沉默和DNA复制，之后我转到科罗拉多大学汤姆·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，特别是利用切赫实验室和CU-Boulder的互动RNA社区的专业知识，了解更多关于RNA的知识。我的研究涉及端粒酶，它复制真核染色体的末端，由一个RNA和几个蛋白质亚基组成。端粒酶在癌症等人类疾病中发挥着重要作用。在我的博士后工作中，我已经开发了1157-NT酵母端粒酶RNA的二级结构模型TLC1，并开发了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体中RNA支架的灵活性，并开始确定重要辅助蛋白在酶机制中的作用。在特定的目标1中，我计划调查酵母端粒酶RNA功能的结构要求，部分是通过移动其结合部位并测试功能来确定Ku是否被RNA灵活地支架。我还将测试TLC1 RNA大小的绝对限制和提出的关于端粒酶RNA二级结构的共识。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。特定目的2部分描述了对酵母端粒酶核心酶在体外是非进行性的假设，因为在体内缺乏反复招募和从其底物中移除该酶的因素。已经开始与其他实验室合作，以获得必要的纯化蛋白，以便在检测中进行测试。最后，在特定的目标3中，我计划检查和开发表达微型化端粒酶RNA Mini-T的细胞的表型，并建议表征我已经确定的相关抑制因子。通过检验这些假设，通过课程作业和小型休假学习新的研究技能，以及获得教学学位和其他相关培训，我希望实现我的目标，即为随后担任教职做好准备。 相关性：端粒酶是一种必须仔细调节才能正常发挥作用的酶，否则它可能会在导致癌症等疾病中发挥关键作用。由于异常过量的端粒酶被认为是~90%的人类癌细胞无限增殖的关键，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，我描述了推断酵母端粒酶功能的最低要求的实验，这是一个在遗传和生物化学上具有优势的系统，有望继续阐明人类酶的特征。