title: Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics.
abstract: The ribonucleoprotein telomerase is an RNA-dependent DNA polymerase that catalyzes the repetitive addition of a short, species-specific, DNA sequence to the ends of linear eukaryotic chromosomes. The single RNA component of telomerase contains both the template sequence for DNA synthesis and a functionally critical pseudoknot motif, which can also exist as a less stable hairpin. Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin-pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments. The urea dependence of these experiments aids in determination of the folding kinetics and thermodynamics. The wild-type pseudoknot behavior is compared and contrasted to a mutant pseudoknot sequence implicated in a genetic disorder-dyskeratosis congenita. These findings clearly identify that this 2nt noncomplementary mutation destabilizes the folding of the wild-type pseudoknot by substantially reducing the folding rate constant (≈ 400-fold) while only nominally increasing the unfolding rate constant (≈ 5-fold). Furthermore, the urea dependence of the equilibrium and rate constants is used to develop a free energy landscape for this unimolecular equilibrium and propose details about the structure of the transition state. Finally, the urea-dependent folding experiments provide valuable physical insights into the mechanism for destabilization of RNA pseudoknots by such chemical denaturants.
authors.affiliation: JILA, University of Colorado and National Institute of Standards and Technology , and ‡Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309-0440, United States.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Holmstrom
authors.forename: David J
authors.initials: DJ
authors.lastname: Nesbitt
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D061925:Immobilized Nucleic Acids / Q000737:chemistry; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000737:chemistry*; D019098:Telomerase / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry*
chemical_list: D061925:Immobilized Nucleic Acids; C422952:telomerase RNA; D012313:RNA; D014508:Urea; D019098:Telomerase