Folding Mechanisms of RNA Pseudoknots Unveiled with Laser Temperature-Jump and Global Modeling Approaches

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes, are determined by their structure, stability and dynamics. Therefore, it is important to elucidate the free energy landscape underlying their thermodynamics/folding mechanisms. Although computational studies have revealed folding pathways and energy landscapes for pseudoknots, experimental measurements to validate them are lacking. This study examines the folding/unfolding of VPK -a variant of the Mouse Mammary Tumor Virus PK involved in ribosomal frameshifting- using fluorescent nucleotide analogs placed at different stem/loop positions as local probes of the RNA folded conformations. We measure the folding kinetics using laser temperature-jump approaches and use global analysis based on a master equation to analyze in a self-consistent way the thermodynamics and kinetics over a broad (20-90 °C) temperature range. These measurements provide the first experimental observation of parallel folding pathways of a pseudoknot and an important benchmark for validating coarse-grained simulations of RNA. Our results, in remarkable agreement with simulations from the Thirumalai group (U. of Texas Austin), demonstrate how the flux between alternative folding pathways of VPK are modulated by ionic strength, with one dominant folding pathway observed at 50 mM KCl, and a parallel pathway emerging at higher ionic strength. These studies highlight a connection between salt-dependent stability of partially folded, intermediate states and folding heterogeneity of pseudoknots. Additionally, in collaboration with the Liang group (UIC Bioengineering), we investigated the thermodynamics of the individual hairpins that make up VPK. The Liang group performed simulations that enumerated all possible folded and misfolded states of these hairpins on a lattice; predictions from the simulations were tested experimentally by designing appropriate mutational studies. We find that even these simple hairpin structures require a minimum of three states to describe their thermodynamics, providing evidence for “off-pathway” traps in the folding energy landscape of VPK. Altogether, this study establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations. Such studies are potentially useful in the design of functional RNA molecules.