Single-MoleculeFluorescence Resonance Energy TransferStudies of the Human Telomerase RNA Pseudoknot: Temperature-/Urea-DependentFolding Kinetics and Thermodynamics.

The ribonucleoprotein telomeraseis an RNA-dependent DNA polymerasethat catalyzes the repetitive addition of a short, species-specific,DNA sequence to the ends of linear eukaryotic chromosomes. The singleRNA component of telomerase contains both the template sequence forDNA synthesis and a functionally critical pseudoknot motif, whichcan also exist as a less stable hairpin. Here we use a minimal versionof the human telomerase RNA pseudoknot to study this hairpin–pseudoknotstructural equilibrium using temperature-controlled single-moleculefluorescence resonance energy transfer (smFRET) experiments. The ureadependence of these experiments aids in determination of the foldingkinetics and thermodynamics. The wild-type pseudoknot behavior iscompared and contrasted to a mutant pseudoknot sequence implicatedin a genetic disorder–dyskeratosis congenita. These findingsclearly identify that this 2nt noncomplementary mutation destabilizesthe folding of the wild-type pseudoknot by substantially reducingthe folding rate constant (≈ 400-fold) while only nominally increasingthe unfolding rate constant (≈ 5-fold).Furthermore, the urea dependence of the equilibrium and rate constantsis used to develop a free energy landscape for this unimolecular equilibriumand propose details about the structure of the transition state. Finally,the urea-dependent folding experiments provide valuable physical insightsinto the mechanism for destabilization of RNA pseudoknots by suchchemical denaturants. [ABSTRACT FROM AUTHOR]