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.
