In vitro preparation and crystallization of vertebrate telomerase subunits

Jing Huang, Christopher J. Bley, Dustin P. Rand, Julian Chen, Ming Lei

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages161-169
Number of pages9
Volume1587
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1587
ISSN (Print)10643745

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Keywords

  • CR4/5
  • Ribonucleoprotein complex
  • Telomerase
  • TRBD
  • X-ray crystallography

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Huang, J., Bley, C. J., Rand, D. P., Chen, J., & Lei, M. (2017). In vitro preparation and crystallization of vertebrate telomerase subunits. In Methods in Molecular Biology (Vol. 1587, pp. 161-169). (Methods in Molecular Biology; Vol. 1587). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6892-3_16