Investigating Telomerase Mechanism by Exploring the RNA

通过探索 RNA 研究端粒酶机制

• 批准号: 7616611
• 负责人: David Clifford Zappulla
• 金额: $ 24.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616611
• 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; 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

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.

在我和罗尔夫博士论文研究期间研究了转录沉默和DMA复制之后， Sternglanz在斯托尼布鲁克大学，我搬到汤姆·切赫在科罗拉多大学的实验室， 博士后工作我的目标一直是加深我的生物化学知识，特别是学习更多 关于RNA，利用Cech实验室和CU-Boulder互动RNA社区的专业知识。我 研究涉及端粒酶，它复制真核细胞染色体的末端， RNA以及几种蛋白质亚基。端粒酶在人类疾病中起着重要作用， 癌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，TLC 1和第一个体外重组酵母端粒酶活性测定。我现建议 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 明确了重要的辅助蛋白在酶作用机制中的作用。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，部分通过确定Ku是否灵活地 通过移动其结合位点，然后测试功能性，我也将测试绝对 TLC 1 RNA大小的限制和端粒酶RNA二级结构的建议共识。我 职业发展计划包括学习如何通过计算方法预测RNA结构， 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 核心酶在体外是非加工性的，因为反复地从其上募集和除去酶的因素 不存在体内底物。已与其他实验室合作，以获得必要的 纯化的蛋白质以在测定中测试。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，并提出表征相关抑制因子 我已经确认过了通过测试这些假设和学习新的研究技能， 课程和一个小休假，以及获得教学学位和其他相关培训，我 我期望达到我的目标，即为以后担任教职做好准备。 相关性：端粒酶是一种酶，必须仔细调节才能适当地发挥作用，否则它会导致 在引发癌症等疾病方面起着关键作用。因为端粒酶的异常过度产生 端粒酶被认为对~90%的人类癌细胞的无限增殖至关重要，端粒酶是一种有吸引力的 药物靶点，虽然一直很难找到合适的抑制剂，因此更多的信息 关于端粒酶功能的研究是必要的。在这里，我描述了一些实验来推导出 酵母端粒酶功能，一个遗传和生物化学上有利的系统，预计将 继续阐明人类酶的特征。