Mechanical unfolding of RNA hairpins

Abstract

Mechanical unfolding trajectories, generated by applying constant force in optical-tweezer experiments, show that RNA hairpins and the P5abc subdomain of the group I intron unfold reversibly. We use coarse-grained Go-like models for RNA hairpins to explore forced unfolding over a broad range of temperatures. A number of predictions that are amenable to experimental tests are made. At the critical force, the hairpin jumps between folded and unfolded conformations without populating any discernible intermediates. The phase diagram in the force-temperature (f, T) plane shows that the hairpin unfolds by an all-or-none process. The cooperativity of the unfolding transition increases dramatically at low temperatures. Free energy of stability, obtained from time averages of mechanical unfolding trajectories, coincides with ensemble averages, which establishes ergodicity. The hopping time between the native basin of attraction (NBA) and the unfolded basin increases dramatically along the phase boundary. Thermal unfolding is stochastic, whereas mechanical unfolding occurs in ''quantized steps'' with great variations in the step lengths. Refolding times, upon force quench, from stretched states to the NBA are at least an order of magnitude greater than folding times by temperature quench. Upon force quench from stretched states, the NBA is reached in at least three stages. In the initial stages, the mean end-to-end distance decreases nearly continuously, and there is a sudden transition to the NBA only in the last stage. Because of the generality of the results, we propose that similar behavior should be observed in force quench refolding of proteins.U nraveling the complexity of the energy landscape of RNA molecules requires exploration of their assembly and unfolding over a wide range of external conditions. In the last decade, a combination of experiments, theoretical arguments, and simulations have been used to decipher the folding mechanisms of RNA molecules (1-3). These studies have shown that RNA folding depends critically on a number of factors, including valence and shape of counterions (4) as well as temperature. Somewhat more surprising, recent experiments have shown that the folding mechanisms depend sensitively on the initial folding conditions (5). In conventional experiments, the difficult-tocharacterize unfolded conformations are typically generated by elevated temperature or by lowering the counterion concentration. In contrast, well defined and vastly different initial conditions can be realized by applying force. Indeed, in remarkable experiments, Bustamante and coworkers (6, 7) have generated mechanical unfolding trajectories for RNA hairpins and Tetrahymena thermophila ribozyme. These experiments, which used constant external force to denature folded RNA, show that unfolding involves multiple routes in which a number of kinetic intermediates are sampled in the transition from the folded state to a stretched conformation (6, 7). The lifetimes of the intermediates vary considerably, which is indicative ...

Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures

“…Although the coarse-grained (CG) models have a number of limitations, they have been proved useful in deciphering the key features that control the folding of a variety of RNA molecules (27,(37)(38)(39)(40). Comparisons of our simulations of RNA pseudoknots to previously observed equilibrium optical and calorimetric melting profiles show excellent agreement.…”

“…Simulations using the Three Interaction Site (TIS) model for RNA (27) compare well with the available experimentally determined thermodynamic transitions. The folding landscapes and kinetics reveal the hidden complexity of RNA folding that manifests itself in the form of parallel folding routes.…”

Determination of thermodynamics and kinetics of RNA reactions by force

“…Therefore, we will expect different apparent kinetics for the bulk methods and the single-molecule force method. Recently, detailed folding kinetic schemes for a small hairpin under various temperatures and forces have been discussed (Hyeon & Thirumalai, 2005.…”

Scaling of hysteresis loop of interacting polymers under a periodic force

“…For example, local motion, which involves atomic fluctuation, side chain motion and loop motion, occurs in the length scale of 0.01 to 5Å and the time involved in such a process is of the order of 10 −15 to 10 −12 s. The motion of helix and protein domain belong to the rigid body motion, whose typical length scales are in between 1 to 100Å and time involved in such motion is in between 10 −9 to 10 −6 s. Here, our interest is in the large-scale motion e.g. helix-coil transition, folding-unfolding transition of proteins and coilglobule transition in polymer, which occurs in the length scale more than 5Å and time involved is about 10 −7 to 100 s. Since, such a time scale is not amenable computationally, therefore, we consider a coarse-grained model of a linear polymer chain and impose restrictive interaction among monomers in such a way that it captures some essential properties of different bio-polymers [39][40][41][42][43]. We follow Ref.…”

“…The native topologybased model, turns out to be quite helpful in predicting the mechanism involved in the DNA unzipping and protein unfolding. It also allowed to decipher the free-energy landscapes of bio-polymers [39][40][41][42][43]49].…”