Programmed ribosomal frameshifting (PRF) in HIV-1 is thought to be stimulated by a hairpin in the mRNA, although a pseudoknot-like triplex has also been proposed. Because the conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, we used optical tweezers to apply tension to the HIV stimulatory structure and monitor its unfolding and refolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterization. Unexpectedly, whereas unfolding reflected the simple two-state behavior typical of many hairpins, refolding was more complex, displaying significant heterogeneity. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. Measuring a variant mRNA containing only the sequence required to form the proposed triplex, it behaved largely in the same way. Nonetheless, very rarely, high-force unfolding events characteristic of pseudoknot-like structures were observed. The rare occurrence of the triplex suggests that the hairpin is the functional stimulatory structure. The unusual heterogeneity of the hairpin dynamics under tension suggests a possible functional role in PRF similar to the dynamics of other stimulatory structures.

draw the T2 RNA back, the re-folded RNA must be unfolded prior to returning. Importantly, the detected unfolding time (stability marker) can clearly report the identity of each folding state, from the free chain to the hairpin, and to the final pseudoknot. The time-dependent occurrence probabilities of all the folding states help us to map the entire stepwise folding pathway in the time scale from 1 second to several minutes. This RNA single-molecule folding approach would lead to broad applications in detecting RNA structuredetermined biological functionalities and their regulation by ligands.