In Vitro Preparation and Crystallization of Vertebrate Telomerase Subunits.
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.
National State Key laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 333 Haike Road, Shanghai, 201210, China. huangjing@sibcb.ac.cn.|Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, 201204, China. huangjing@sibcb.ac.cn.
Jing
J
Huang
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287, USA.
Christopher J
CJ
Bley
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287, USA.
Dustin P
DP
Rand
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287, USA.
Julian J L
JJ
Chen
National State Key laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 333 Haike Road, Shanghai, 201210, China. leim@sibcb.ac.cn.|Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, 201204, China. leim@sibcb.ac.cn.|School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai, 201210, China. leim@sibcb.ac.cn.
Ming
M
Lei
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D002875:Chromosomes / Q000235:genetics; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D056890:Eukaryota / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D012313:RNA / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D014714:Vertebrates / Q000235:genetics*
D012313:RNA; D019098:Telomerase
CR4/5; Ribonucleoprotein complex; TRBD; Telomerase; X-ray crystallography