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Real-Time Control of the Energy Landscape by Force Directs the Folding of RNA Molecules

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Real-Time Control of the Energy Landscape by Force Directs the Folding of RNA Molecules Real-time control of the energy landscape by force directs the folding of RNA molecules Pan T. X. Li*†§, Carlos Bustamante*¶ʈ, and Ignacio Tinoco, Jr.*§ Departments of *Chemistry and ¶Physics and Molecular and Cell Biology, ʈHoward Hughes Medical Institute, University of California, Berkeley, CA 94720 Contributed by Ignacio Tinoco, Jr., March 9, 2007 (sent for review January 30, 2007) The rugged folding-energy landscapes of RNAs often display many a competing minima. How do RNAs discriminate among competing b G G U G C A conformations in their search for the native state? By using optical laser trap C G FF G C FU A U tweezers, we show that the folding-energy landscape can be G C U manipulated to control the fate of an RNA: individual RNA mole- C Handle A UA U G C cules can be induced into either native or misfolding pathways by XF RNA A U C G C G modulating the relaxation rate of applied force and even be Handle B U A X G C A U redirected during the folding process to switch from misfolding to U A U A G C native folding pathways. Controlling folding pathways at the G U U A single-molecule level provides a way to survey the manifold of C G U G C G folding trajectories and intermediates, a capability that previously G C 5'- G C -3' was available only to theoretical studies. micropipette TAR mechanical force ͉ misfolding ͉ optical tweezers ͉ RNA folding ͉ c single-step multiple-steps misfolding single molecule start 20 rip rip rip ecroF )Np( ecroF )Np( he folding of a macromolecule can be thought of as a biased s e t 15 a diffusion over an energy surface that describes the thermo- i T d e dynamic and kinetic constraints of possible intramolecular in- mr 10 zip etn rescue teractions. RNAs differ from proteins in the nature, strength, i ec r specificity, and degeneracy of the interactions that stabilize their zip oF 5 native structures. Whereas proteins are made of 20 amino acids, 20 nm it takes only 4 nucleotides to build RNAs. This simpler compo- 0 sition, endowed with robust base-pairing rules, greatly increases Extension the promiscuity of interactions between any given nucleotide and Fig. 1. Optical-tweezers assay for studying folding, misfolding, and rescue of the rest of the RNA structure. These RNA–protein differences TAR RNA. (a) Experimental design. RNA hairpin TAR, flanked by dsDNA/RNA are reflected in the topography of the energy surface over which handles, is tethered to two microspheres (11). One microsphere is held by a the molecules diffuse in their search for the native structure: the force-measuring trap, and the other is held on a micropipette. By moving the energy surfaces of RNAs are significantly more rugged than micropipette, tension (F) is exerted on the molecule, and the change in those of proteins, containing many competing local minima extension (X) is measured (13). (b) Native structure of TAR. (c) Three types of (1–4). Even relatively small RNAs, such as tRNA (4–6), tend to force-extension curves for TAR RNA. In each experiment, force was first relaxed from 20 to 1 pN (red) and then was raised back to 20 pN (blue). fold into stable alternate secondary structures with energies only ϭ Unfolding is indicated by rips that increase the extension, whereas folding and a few kilocalories (1 kcal 4.18 kJ on first use) per mole apart rescue are indicated by zips that shorten the extension. The arrows point to (4). Once trapped kinetically in a secondary structure with the fluctuations/intermediates on refolding. suboptimal folding energy, it is difficult for an RNA molecule to reach its native structure (1). The promiscuous nature of secondary interactions in RNA begs loading and unloading rate, respectively). Significantly, the applied answers for three questions: How do RNAs find their native tension slows the hairpin folding kinetics from an order of 106 sϪ1 structure? How do they avoid the kinetic traps on their rugged at zero force (14) to a more measurable range from 102 to 10Ϫ2 sϪ1 folding-energy landscape? And, how can RNAs switch between at its transition force such that the process of the folding can be alternate structures in response to changes in the cellular environ- monitored in real time. The force (F)-dependent un/refolding rate ment, as happens, for example, during transcription attenuation in constant (kF) can be described by an Arrhenius-type equation (15): bacteria (7)? To address these questions, we need to follow the ϭ ϩ ‡͞ folding trajectories of RNA molecules as they diffuse over their ln kF ln k0 FX kBT, [1] energy landscape in search of stable conformations. Because of the where k0 is the apparent rate constant of the mechanical structural polymorphism and manifold pathways of RNA, single- unfolding process at zero force, and k T is Boltzmann’s constant molecule approaches (8–12) are particularly useful for answering B these questions by identifying folding intermediates and distinguish- BIOPHYSICS ing native pathways from those that misfold. Author contributions: P.T.X.L., C.B., and I.T. designed research; P.T.X.L. performed research; Here we use optical tweezers to unfold and control the refolding P.T.X.L. and I.T. analyzed data; and P.T.X.L., C.B., and I.T. wrote the paper. of single RNA molecules by force (11, 13). Two micrometer-sized The authors declare no conflict of interest. beads are attached to the ends of an RNA molecule and are Abbreviation: TAR, transactivation response region. manipulated by optical tweezers to exert force on the RNA (Fig. †Present address: Department of Biological Sciences, University at Albany, State University 1a). The unfolding and refolding of the RNA molecule, induced by of New York, Albany, NY 12222. controlled application of force, is monitored by nanometer changes §To whom correspondence may be addressed. E-mail: [email protected] or panli@ in the end-to-end distance of the molecule. In a typical ‘‘pulling’’ albany.edu. experiment, force is increased or decreased at a fixed rate (the © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702137104 PNAS ͉ April 24, 2007 ͉ vol. 104 ͉ no. 17 ͉ 7039–7044 Downloaded by guest on September 27, 2021 60 a 40 b 35 50 30 e %ded 40 c n 25 er l r 30 20 o f uc siM cO 15 20 10 10 5 0 0 3 4 5 6 7 8 9 10 11 12 15 20 25 30 35 40 number of ss nucleotides paired Unloading Rate (pN/s) c 18 d 2 16 1.5 14 1 ])U/1( nl r[ nl r[ nl ])U/1( ecnerruccO 12 0.5 10 0 8 6 -0.5 4 -1 2 -1.5 0 -2 5 6 7 8 9 10 11 12 13 8 8.5 9 9.5 10 10.5 11 Rescue Force (pN) Rescue Force (pN) Fig. 2. Misfolding and rescue of TAR RNA. (a) Histogram of the decrease in end-to-end distance, ⌬Xrescue, in the rescue. ⌬Xrescue was converted to the number of single-stranded nucleotides that become paired in the rescue. (b) Fraction of misfolded trajectories as a function of unloading rates. Between 122 and 324 total trajectories were collected at each unloading rate. (c) Histogram of the rescue forces in the force-ramp experiment at an unloading rate of 11.2 pN/s followed by a loading rate of 2 pN/s. The rescue force was defined as the force at which the rescue transition starts. (d) The rescue-force distribution in c is fitted to Eq. ‡ Ϯ 2, yielding an Xrescue1 of 6 1 nm. times temperature in Kelvin. X‡, the distance to the transition at Ϸ13–16 pN. In contrast, when the tension on the molecule was state, is positive for unfolding and negative for refolding. Hence, allowed to relax at a rate of 1.5 pN/s, Ͼ95% of the refolding curves we can control the un/refolding rate constants by manipulating show multiple-step refolding, displaying a decreasing extension with the tension on a molecule. characteristic fluctuations (Fig. 1c, black arrows) followed by a When an RNA molecule is pulled and relaxed very slowly such small zip. This zip corresponds to the folding of an average of 26 nt, that the process is thermodynamically reversible, simple hairpins which is half the value for the refolding of the entire molecule. are often seen to unfold and refold cooperatively in a single step Subsequent pulling of such molecules displayed unfolding charac- (11, 16–21). One example is the 52-nt HIV transactivation teristics similar to those observed in the slow relaxation curves, response region (TAR) RNA (22), which folds into a 21-bp indicating that these RNAs had folded into the native structure. The hairpin with a 3-nt bulge (Fig. 1b) (23). The equilibrium force back-and-forth oscillations in the force-extension curve before the zip are likely successive refolding and unfolding events as the (F1/2), at which its unfolding and refolding rates are equal, is 12.4 pN in 100 mM KCl, pH 8, at 22°C (19). When the force is held molecule moves into and out of alternative, competing folding pathways. Once an RNA molecule, following this search process, constant at or near the F1/2 value, an individual TAR molecule can be seen to transit between folded and unfolded states folds into intermediate structures that contain only native contacts, without detectable intermediates.
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