Folding in Human Telomerase RNA Pseudoknots: Kinetic and Thermodynamic Studies via Single-Molecule FRET

Abstract

Telomerase is a ribonucleoprotein (RPN) responsible for maintaining the ends of linear eukaryotic chromosomes and has serious implications for both aging and cancer. The single RNA component of the RNP enzyme has a highly conserved pseudoknot motif that is critical for proper biological function. Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) microscopy is used to investigate the folding kinetics and thermodynamics of a minimal wild-type (WT) pseudoknot construct. Urea is used in conjunction with the smFRET experiments to: (i) aid in determination of the kinetic and thermodynamics parameters and (ii) develop a qualitative picture for the folding landscape. These results are compared and contrasted to the results of a nearly identical RNA construct with a 2 nt substitution mutation associated with the genetic disorder dyskeratosis congentia (DKC). As expected, the WT RNA construct (ΔG°WT = −4.2 ± 0.2 kcal/mol) is substantially more stable than the DKC construct (ΔG°DKC = 0.26 ± 0.05 kcal/mol]. The kinetic origin of this differential stability is the result of a substantially increased folding rate constant (∼400 times faster) for the WT and a subtle reduction of the unfolding rate constant (∼5 times slower).View Large Image | View Hi-Res Image | Download PowerPoint Slide