Abstract
Telomerase is a unique reverse transcriptase that replicates the telomeric DNA at most eukaryotic chromosomal ends. The telomerase consists of the catalytic protein subunit TERT and the RNA component TR that provides the template for telomeric DNA synthesis. In vitro reconstitution of telomerase core components in large quantity is the prerequisite to studying the catalytic mechanisms of telomerase at the structural level; however, large-scale preparation of recombinant telomerase, especially that of higher eukaryotes, has been a big challenge for a long time. It has been known that the CR4/5 domain of the vertebrate TR binds to the TRBD domain of TERT and the interaction is essential to the assembly and enzymatic activity of telomerase. We assembled the TRBD-CR4/5 ribonucleoprotein complex of the medaka fish telomerase in vitro and determined its atomic structure through X-ray crystallography. Our study provides the structural insight into the RNA-protein recognition mechanism that is common to most eukaryotic telomerase. The methods of our study are also applicable to large-scale preparations of other ribonucleoprotein complexes for structural studies.
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Acknowledgments
We thank Y. Li (University of Michigan) and K. Wan (University of Michigan) for technical support and F. Guo (University of California, Los Angeles) for suggestions. This work is supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08010201 to M.L.), the Ministry of Science and Technology of China (2013CB910402 to M.L.), the National Natural Science Foundation of China (31330040 and 31525007 to M.L.), National Science Foundation of China (31570766 and U1632130 to J.H.), the Ministry of Science and Technology of China (2016YFA0501800 to J.H.) and Shanghai Pujiang Talents Project (15PJ1409300 to J.H.).
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Huang, J., Bley, C.J., Rand, D.P., Chen, J.J.L., Lei, M. (2017). In Vitro Preparation and Crystallization of Vertebrate Telomerase Subunits. In: Songyang, Z. (eds) Telomeres and Telomerase. Methods in Molecular Biology, vol 1587. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6892-3_16
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DOI: https://doi.org/10.1007/978-1-4939-6892-3_16
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