NDLI: In vitro reconstitution of the active T. castaneum telomerase.

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In vitro reconstitution of the active T. castaneum telomerase.

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In vitro reconstitution of the active T. castaneum telomerase.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Schuller, Anthony P. Harkisheimer, Michael J. Skordalakes, Emmanuel
Description	Country affiliation: United States Author Affiliation: Schuller AP ( Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania, USA.)
Abstract	Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro. Telomerase is a specialized reverse transcriptase that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit. Additionally, telomerase is dormant in most somatic cells in adults, but is active in cancer cells where it promotes cell immortality. The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres. Prior to 2008, only structures for individual telomerase domains had been solved. A major breakthrough in this field came from the determination of the crystal structure of the active, catalytic subunit of T. castaneum telomerase, TcTERT. Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/2799
Journal	Journal of Visualized Experiments
Issue Number	53
Language	English
Publisher	MyJove Corp.
Publisher Date	2011-07-14
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Telomerase Metabolism Tribolium Enzymology Animals Catalytic Domain Recombinant Proteins Biosynthesis Chemistry Genetics Ribonucleoproteins Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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