title: In vitro reconstitution of the active T. castaneum telomerase.
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.
authors.affiliation: Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania, USA.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Schuller
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Harkisheimer
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Skordalakes
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism; D019098:Telomerase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014231:Tribolium / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D019098:Telomerase