Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

Abstract

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at ∼10 and ∼5 piconewtons (pN), with ensemble rate constants of ∼0.1 sec⁻¹, by stepwise force–drop experiments. Folding studies of the isolated 5′-hairpin construct suggested that the 5′-hairpin within the pseudoknot forms first, followed by formation of the 3′-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of ∼46 pN, at an average rate of ∼0.05 sec⁻¹. In the constant-force folding trajectories at ∼10 pN and ∼5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.

Keywords

• single-molecule
• optical tweezers
• telomerase RNA pseudoknot
• folding pathways
• unfolding/folding kinetics
• force–jump/drop