title: Functional importance of telomerase pseudoknot revealed by single-molecule analysis.
abstract: Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Mariana
authors.initials: M
authors.lastname: Mihalusova
authors.forename: John Y
authors.initials: JY
authors.lastname: Wu
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhuang
mesh_terms: D001483:Base Sequence; D019921:Biotinylation; D002384:Catalysis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D020035:Holoenzymes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*; D016808:Tetrahymena thermophila / Q000235:genetics
chemical_list: D020035:Holoenzymes; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase