RNA Pseudoknot Folding Energy Landscape Elucidated with T-Jump Measurements and Kinetic Modeling

Abstract

Pseudoknots are minimal tertiary motifs in RNA that are involved in many biological functions determined by their structure, stability and dynamics. They also stabilize the structures of several ribozymes and are often the rate-limiting step in their folding pathways. Although many studies have reported on pseudoknot stability, and theoretical and computational studies have proposed folding pathways for some pseudoknots, there are few experimental studies on pseudoknot folding kinetics. Therefore, a complete picture of how pseudoknots fold to achieve their functional structure is lacking.Here, we report folding kinetics of the VPK pseudoknot, a variant of the Mouse Mammary Tumor Virus (MMTV) pseudoknot, which is involved in ribosomal frameshifting. We combine rapid temperature-jump (T-jump) with time-resolved fluorescence spectroscopy, and global analysis of equilibrium and kinetics measurements, to elucidate the folding energy landscape. We use 2AP (a fluorescent analog of adenine) placed at different positons along the RNA sequence as a probe of RNA conformations, both under equilibrium conditions and in response to the T-jump perturbation. We also, independently, measure the equilibrium and folding kinetics of two hairpin structures that are believed to be intermediates in the folding pathways. The complete set of equilibrium and kinetics measurements on the pseudoknot and the two hairpins are described in a self-consistent manner in terms of a 4-state kinetic model and a minimal set of parameters. Our results provide the first experimental evidence of multiple parallel folding/unfolding pathways in RNA pseudoknots, as indicated by previous simulations studies.