Which steps in the folding process of the native hairpin are perturbed by the fast force relaxation? High force favors the longer single-strand forms over base-paired structures. Such forces were needed to break the nonnative interactions that trapped the RNA structure, allowing the molecule to search for its correct folding pathway and effectively rescuing it from the misfolded state. If they were so, one would expect them to fold quickly at 6 pN are required for their conversion to the native hairpin. The low threshold of the transition force argues against the idea that the prerescue structures are on-pathway folding intermediates. Rescue occurred between 6 and 12 pN at a loading rate of 2 pN/s ( Fig. The faster the force is decreased, the more likely a molecule becomes trapped into a variety of stable misfolded structures with less base pairs and longer end-to-end distances than the native hairpin, as shown by the increase in frequency of misfolding with increasing force relaxation rates ( Fig. Accordingly, we term this zip a “rescue” transition. We conclude that the structures at low force are misfolded, and that the application of force allowed the RNA molecules to form the native hairpin in the zip transition. As the force was further raised, the force-extension curve always showed a rip similar to the unfolding of the native hairpin. 2 a), indicating that a range of partially folded structures existed before the transition. The magnitude of this zip shows a broad distribution with a mean of ≈30 single-stranded nucleotides becoming paired ( Fig. This transition, not observed previously, is a refolding event in that the extension of the molecule is shortened, yet it occurs as the force is increasing, which normally causes unfolding. When the force on the molecule was subsequently increased, the extension of the RNA increased monotonically, retracing the relaxation curve until a zip transition suddenly shortened the end-to-end distance of the molecule, against the increasing force. Once an RNA molecule, following this search process, folds into intermediate structures that contain only native contacts, the remaining helix can form cooperatively, as manifested by the observed zip.Īt unloading rates >2 pN/s, we observed another type of relaxation curve that showed no zipping at all at low force the extension of the RNA never returned to that of the native hairpin ( Fig. The back-and-forth oscillations in the force-extension curve before the zip are likely successive refolding and unfolding events as the molecule moves into and out of alternative, competing folding pathways. Subsequent pulling of such molecules displayed unfolding characteristics similar to those observed in the slow relaxation curves, indicating that these RNAs had folded into the native structure. This zip corresponds to the folding of an average of 26 nt, which is half the value for the refolding of the entire molecule. 1 c, black arrows) followed by a small zip. In a typical cycle, the force was first ramped down from 20 to 95% of the refolding curves show multiple-step refolding, displaying a decreasing extension with characteristic fluctuations ( Fig. ![]() To explore the possibility of alternative folding pathways, individual TAR molecules were stretched and relaxed repeatedly at approximately fixed loading and unloading rates (pN/s). Once trapped kinetically in a secondary structure with suboptimal folding energy, it is difficult for an RNA molecule to reach its native structure ( 1). Even relatively small RNAs, such as tRNA ( 4– 6), tend to fold into stable alternate secondary structures with energies only a few kilocalories (1 kcal = 4.18 kJ on first use) per mole apart ( 4). These RNA–protein differences are reflected in the topography of the energy surface over which the molecules diffuse in their search for the native structure: the energy surfaces of RNAs are significantly more rugged than those of proteins, containing many competing local minima ( 1– 4). This simpler composition, endowed with robust base-pairing rules, greatly increases the promiscuity of interactions between any given nucleotide and the rest of the RNA structure. Whereas proteins are made of 20 amino acids, it takes only 4 nucleotides to build RNAs. RNAs differ from proteins in the nature, strength, specificity, and degeneracy of the interactions that stabilize their native structures. The folding of a macromolecule can be thought of as a biased diffusion over an energy surface that describes the thermodynamic and kinetic constraints of possible intramolecular interactions.
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