title: Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis.
abstract: Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.|Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Parks
authors.affiliation: Biophysics Program, Stanford University, Stanford, California 94305, USA.
authors.forename: Kalli
authors.initials: K
authors.lastname: Kappel
authors.affiliation: Biophysics Program, Stanford University, Stanford, California 94305, USA.|Department of Biochemistry, Stanford University, Stanford, California 94305, USA.|Department of Physics, Stanford University, Stanford, California 94305, USA.
authors.forename: Rhiju
authors.initials: R
authors.lastname: Das
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.|Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Stone
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D055162:Biocatalysis; D001710:Biotin / Q000737:chemistry; D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D000072760:Single Molecule Imaging; D019809:Streptavidin / Q000737:chemistry; D040681:Structural Homology, Protein; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D012261:Ribonucleoproteins; C422952:telomerase RNA; D012313:RNA; D001710:Biotin; D019809:Streptavidin; C509186:TERT protein, human; D019098:Telomerase
keywords: Rosetta; modeling; polymerase; pseudoknot; single-molecule FRET; telomerase