Structural Studies of Tetrahymena Telomerase

四膜虫端粒酶的结构研究

• 批准号: 1022379
• 负责人: Juli Feigon
• 金额: $ 124.9万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1022379&HistoricalAwards=false
• 关键词: Structural; Studies; Tetrahymena; Telomerase

Intellectual Merit Telomerase is a large, multi-subunit ribonucleoprotein (RNP) complex that plays an essential role in maintenance of telomere DNA at the ends of linear chromosomes in eukaryotes. Telomerases from all species contain an essential RNA component (telomerase RNA, TER), a unique reverse transcriptase protein (telomerase reverse transcriptase, TERT), and various accessory proteins which together are required for assembly, accumulation, localization, telomere repeat addition proccessivity, and catalysis. The telomere repeat sequence, TTGGGG in Tetrahymena and TTAGGG in vertebrates, is synthesized on an RNA template contained in the TER. Telomerase has been the focus of intense study due to its role in preventing chromosomal instability. Due to the low abundance of telomerase in most organisms, it has been difficult to isolate the intact holoenzyme, and the roles of identified protein and RNA components in RNP processing, assembly, and function have only been partially characterized. In spite of the enormous interest in telomerase, to date there are no structures of any RNA-protein complexes of telomerase. The earliest studies on telomerase were done on ciliates, which have many more telomeres and therefore more telomerase than other organisms. The discovery of telomerase in Tetrahymena led to the 2009 Nobel Prize to Elizabeth Blackburn, Carol Greider, and Jack Szostak. Telomerase activity can be reconstituted in vitro from TER and TERT alone, but other proteins are required for function in vivo. The Tetrahymena telomerase holoenzyme has been purified and protein components identified using affinity chromatography from Tetrahymena strains containing affinity tagged TERT. Among these is the holoenzyme assembly protein p65, which together with TERT and TER comprises the catalytic core of Tetrahymena telomerase and is required in vivo for assembly of these components of the holoenzyme. This project focuses on understanding Tetrahymena telomerase holoenzyme assembly and structure, including the role of p65 in Tetrahymena telomerase assembly. NMR, chemical probing, and X-ray crystallography will be used to investigate protein and RNA interactions, and cryoelectron microcroscopy will be used to investigate the overall structure of the Tetrahymena telomerase holoenzyme. The long-range goal is to combine information from solution and crystal structures of components of Tetrahymena telomerase with cryoelectron microscopy images to obtain a detailed understanding of the architecture, assembly and dynamics of this essential macromolecular machine. This work should lead to fundamental new insights into how telomerase functions to regulate telomere length, and ultimately to how changes in telomerase activity affect both cell proliferation and cellular aging. Broader Impacts The structural studies of Tetrahymena telomerase will integrate existing information on the cellular function and biochemistry of this important enzyme complex, and provide new insights into the RNA folding, tertiary structure, role in catalysis, protein and holoenzyme structure, and assembly of the telomerase RNP. As such, this project will impact biology across the full breadth of science. These projects provide essential training for undergraduate and graduate students and postdoctoral fellows in structural biology and biophysics of nucleic acids and nucleic acid-protein complexes. This is an area in which women and minorities have long been underrepresented, and this lab provides a role model for them. As an example of the long-range impact of this work in terms of education, several postdoctoral fellows who have worked on NSF projects in the Feigon lab have gone on to faculty positions. Undergraduate students who have worked on these projects have gone on to graduate school. Another important example of the long-range impact of this work in terms of education is that results from the previous NSF funding cycle have already been published as figures and discussion in two biochemistry and structural biology textbooks. Lessons in structural biology are also incorporated into honors undergraduate biochemistry classes by having the students make a web-based CHIME (utilizing html and rasmol) structural demonstration of a nucleic acid or a nucleic acid-protein complex. These and some simpler CHIME demos (available on the Feigon lab web page and Virtual Office Hours) can be used as structure teaching tools by faculty for undergraduate and graduate biochemistry core courses, including teaching about telomerase. Future plans also include outreach to local high school students through programs in the California Nanosystems Institute at UCLA.

智力优先型端粒酶是一种大型的多支亚基核糖核蛋白（RNP）复合物，在真核生物中线性染色体的末端维持端粒DNA中起着至关重要的作用。来自所有物种的端粒酶都包含必需的RNA成分（端粒酶RNA，TER），一种独特的逆转录酶蛋白（端粒酶逆转录酶，TERT）和各种辅助蛋白，这些蛋白共同需要组装，积累，本地化，端粒重复添加添加性添加性添加性和催化。在TER中包含的RNA模板上合成了端粒重复序列，四膜虫中的TTGGGG和脊椎动物中的Ttaggg。由于端粒酶在预防染色体不稳定性中的作用，端粒酶一直是激烈研究的重点。由于大多数生物体中端粒酶的丰度低，因此很难分离完整的全酶，并且仅部分表征了鉴定蛋白和RNA成分在RNP加工，组装和功能中的作用。尽管对端粒酶具有巨大的兴趣，但迄今为止，端粒酶的任何RNA蛋白质复合物都没有结构。对端粒酶的最早研究是对纤毛的，它们的端粒比其他生物更重要，因此端粒酶更多。在四氢烷中发现端粒酶的发现导致2009年诺贝尔奖获得了伊丽莎白·布莱克本，卡罗尔·格雷德和杰克·斯佐斯塔克。端粒酶活性可以单独从TER和TERT中重新构造，但是在体内功能需要其他蛋白质。四心肌端粒酶全酶已被纯化，并使用含有亲和力标记的TERT的四膜菌菌株的亲和色谱鉴定蛋白质成分。其中包括全酶组装蛋白p65，它与Tert和Ter一起包含四膜烷端粒酶的催化核心，并且在体内需要用于组装全酶的这些成分。该项目的重点是理解四氢烷端粒酶全酶的组装和结构，包括p65在四氢烷端粒酶组装中的作用。 NMR，化学探测和X射线晶体学将用于研究蛋白质和RNA相互作用，而冷冻电子微序镜将用于研究四氢烷端酶全酶的整体结构。远程目标是结合四膜烷端粒酶组件的溶液和晶体结构与冷冻电子显微镜图像，以获得对这款基本大分子机器的体系结构，组装和动态的详细理解。这项工作应导致对端粒酶功能如何调节端粒长度的基本新见解，并最终导致端粒酶活性的变化如何影响细胞增殖和细胞衰老。更广泛的影响四氢烷端粒酶的结构研究将整合有关该重要酶复合物的细胞功能和生物化学的现有信息，并为RNA折叠，第三纪结构，在催化，蛋白质和全蛋白质结构中的作用以及端粒酶RNP的组装提供新的见解。因此，该项目将影响整个科学广度的生物学。这些项目为本科和研究生以及核酸和核酸 - 蛋白质复合物生物物理学的博士后研究员提供了必不可少的培训。在这个领域，妇女和少数民族长期以来的代表性不足，该实验室为她们提供了榜样。作为这项工作在教育方面的长期影响的一个例子，在Feigon实验室从事NSF项目的几位博士后研究员继续担任教师职位。从事这些项目的本科生已经进入了研究生院。这项工作在教育方面的长期影响的另一个重要例子是，以前的NSF融资周期的结果已经在两个生物化学和结构生物学教科书中作为数字和讨论发表。结构生物学领域的课程还通过让学生制作基于网络的核（利用HTML和Rasmol）结构性证明核酸或核酸 - 蛋白质蛋白复合物的结构表现，将结构生物学的课程纳入荣誉本科生物化学类别。这些和一些更简单的核演示（在Feigon Lab网页和虚拟办公室时间上可用）可以通过教师的本科和研究生生物化学核心课程作为结构教学工具，包括有关端粒酶的教学。未来的计划还包括通过加州大学洛杉矶分校的加利福尼亚纳米系统研究所的计划向当地高中学生推广。