Structural Studies of Tetrahymena Telomerase

智能优点端粒酶是一个大的、多亚单位的核糖核蛋白(RNP)复合体，在维持真核生物线性染色体末端的端粒DNA方面起着至关重要的作用。所有物种的端粒酶都含有一个基本的RNA组分(端粒酶RNA，TER)，一个独特的逆转录酶蛋白(端粒酶逆转录酶，TERT)，以及各种辅助蛋白，这些都是组装、积累、定位、端粒重复序列添加过程和催化所必需的。端粒重复序列，四膜虫中的TTGGGG和脊椎动物中的TTAGGG，是在TER中包含的RNA模板上合成的。由于端粒酶在防止染色体不稳定中的作用，它一直是研究的热点。由于大多数生物中端粒酶的丰度较低，因此很难分离出完整的全酶，而且已识别的蛋白质和RNA组分在RNP加工、组装和功能中的作用还只有部分表征。尽管人们对端粒酶有极大的兴趣，但到目前为止还没有任何端粒酶的RNA-蛋白质复合体的结构。关于端粒酶的最早研究是在纤毛虫身上进行的，纤毛虫的端粒比其他生物多得多，因此端粒酶也更多。四膜虫中端粒酶的发现使伊丽莎白·布莱克本、卡罗尔·格雷德和杰克·绍斯塔克获得了2009年诺贝尔奖。TER和TERT单独在体外可以重组端粒酶活性，但在体内需要其他蛋白质来发挥作用。从含有亲和标签TERT的四膜虫菌株中纯化了四膜虫端粒酶全酶，并用亲和层析法鉴定了其蛋白质组分。其中包括全酶组装蛋白P65，它与TERT和TER一起构成四膜虫端粒酶的催化核心，是体内组装这些全酶成分所必需的。本项目的重点是了解四膜虫端粒酶的全酶组装和结构，包括p65在四膜虫端粒酶组装中的作用。核磁共振、化学探针和X射线结晶学将被用来研究蛋白质和RNA的相互作用，而冷冻电子显微镜将被用来研究四膜虫端粒酶全酶的整体结构。长期目标是将来自四膜虫端粒酶组分的溶液和晶体结构的信息与冷冻电子显微镜图像相结合，以获得对这一重要的大分子机器的结构、组装和动力学的详细了解。这项工作将导致对端粒酶如何发挥作用来调节端粒长度的根本新见解，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。四膜虫端粒酶的结构研究将整合关于这一重要酶复合体的细胞功能和生物化学的现有信息，并为RNA折叠、三级结构、在催化中的作用、蛋白质和全酶结构以及端粒酶RNP的组装提供新的见解。因此，这个项目将影响整个科学领域的生物学。这些项目为本科生、研究生和博士后研究员提供了有关核酸和核酸-蛋白质复合体的结构生物学和生物物理学的基本培训。这是一个长期以来女性和少数族裔代表不足的领域，这个实验室为她们提供了一个榜样。作为这项工作在教育方面的长期影响的一个例子，几名曾在费贡实验室参与NSF项目的博士后研究员已经进入教员职位。参与过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，前一个国家科学基金资助周期的结果已经在两本生物化学和结构生物学教科书中以数字和讨论的形式公布。结构生物学的课程也被纳入本科生物化学荣誉课程，让学生在网上制作一个核酸或核酸-蛋白质复合体的结构演示(利用html和rasmol)。这些和一些更简单的CHINE演示(可在Feigon实验室网页和虚拟办公时间内获得)可作为本科生和研究生生物化学核心课程的结构教学工具，包括教学端粒酶。未来的计划还包括通过加州大学洛杉矶分校的加州纳米系统研究所的项目向当地高中生推广。

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.