Published on Web 01/14/2006
Exploring the Energy Landscape of a Small RNA Hairpin Hairong Ma,† David J. Proctor,‡ Elzbieta Kierzek,⊥ Ryszard Kierzek,⊥ Philip C. Bevilacqua,*,‡ and Martin Gruebele*,†,§ Contribution from the Center for Biophysics and Computational Biology, Department of Chemistry and Department of Physics, UniVersity of Illinois, Urbana, Illinois 61801, Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802, and Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan˜ , Poland Received August 6, 2005; E-mail:
[email protected];
[email protected] Abstract: The energy landscape of a small RNA tetraloop hairpin is explored by temperature jump kinetics and base-substitution. The folding kinetics are single-exponential near the folding transition midpoint Tm. An additional fast phase appears below the midpoint, and an additional slow phase appears above the midpoint. Stem mutation affects the high-temperature phase, while loop mutation affects the low-temperature phase. An adjusted 2-D lattice model reproduces the temperature-dependent phases, although it oversimplifies the structural interpretation. A four-state free energy landscape model is generated based on the lattice model. This model explains the thermodynamics and multiphase kinetics over the full temperature range of the experiments. An analysis of three variants shows that one of the intermediate RNA structures is a stacking-related trap affected by stem but not loop modification, while the other is an early intermediate that forms some stem and loop structure. Even a very fast-folding 8-mer RNA with an ideal tetraloop sequence has a rugged energy landscape, ideal for testing analytical and computational models.
Introduction
The folding of RNA starts from a large ensemble of partially or wholly unfolded conformations and navigates toward the much smaller native ensemble. RNA folding is more hierarchically organized than protein folding.1,2 In particular, RNA readily forms independently stable secondary structure elements at low counterion concentration; thus, the folding of secondary structure motifs can be studied in the absence of tertiary structure formation. The time scale for secondary structure formation (e.g., zipping) can be as fast as the 0.1-1 µs range.3 RNA hairpins consist of a base-paired stem capped with a loop (Figure 1). The hairpin motif is among the most common secondary structure elements in RNA and takes part in various important biological functions including ligand binding and tertiary folding initiation.2,4,5 Although the function, thermodynamics, and kinetics of RNA hairpins have been extensively investigated,2,6-10 our knowledge of the full free energy landscape of RNA hairpins is incomplete. †
Center for Biophysics and Computational Biology, University of Illinois. Department of Chemistry and Department of Physics, University of Illinois. ‡ Department of Chemistry, The Pennsylvania State University. ⊥ Institute of Bioorganic Chemistry, Polish Academy of Sciences. §
(1) Brion, P.; Westhof, E. Annu. ReV. Biophys. Biomol. Struct. 1997, 26, 113137. (2) Tinoco, I.; Bustamante, C. J. Mol. Biol. 1999, 293, 271-281. (3) Poerschke, D. Biophys. Chem. 1974, 2, 97-101. (4) Uhlenbeck, O. C. Nature 1990, 346, 613-614. (5) Varani, G. Ann. ReV. Biophys. Biomol. Struct. 1995, 24, 379-404. (6) Wilson, K. S.; Hippel, P. H. v. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8793-8797. 10.1021/ja0553856 CCC: $33.50 © 2006 American Chemical Society
Figure 1. Structure of the gcUUCGgc hairpin (pdb ID: 1F7Y, truncated to residues 31-38). There are stabilizing hydrogen bonds between the first U and the fourth G in the loop, as well as the third C and a phosphate. Visualization was via VMD.23
In some cases two-state models can be used to describe RNA hairpin-folding kinetics.11,12 However, recent experiments and theoretical calculations suggest that RNA/DNA hairpin kinetics cannot be described generally by two-state models.9,10,13 A rough (7) Bonnet, G.; Krichevsky, O. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 86028606. (8) Shu, Z. Y.; Bevilacqua, P. C. Biochemistry 1999, 38, 15369-15379. J. AM. CHEM. SOC. 2006, 128, 1523-1530
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RNA energy landscape arises from energetic and topological frustration. Energetic frustration is caused by the conflict between base stacking (favoring collapsed states of the molecule) and electrostatic interactions (favoring extended states); topological frustration is caused by backbone connectivity.14 Temperature and mutation can be used to “tune in” different parts of a rough energy landscape. Here we report sequencedependent folding kinetics of an RNA hairpin tetraloop family by using laser temperature-jumps. The sequences of these hairpins are chosen to differ either in the loop or the stem. We find that the hairpin has simple kinetics at the transition midpoint Tm as previously reported,15 but multiphasic kinetics elsewhere. Stem and loop mutations have different effects on the observed multiphasic kinetics at different temperatures. The experiments, coupled with a simple lattice model and a four-state model simulation, provide a structural model for the RNA hairpinfolding mechanism. Materials and Methods
Design of the RNA Hairpin Model System. The 8 nt sequence, a tetraloop with a 2 base pair stem, is the smallest RNA that folds into a stable and well-formed hairpin.16 The short stem with only 2 base pairs was chosen to lower the melting temperature so that fully unfolded conditions could be explored, and to minimize alternative base pairings and the chance of falling into a misfolded trap. The short stem may also help increase the effect of loop composition on folding kinetics. To examine the sequence dependence of hairpin-folding kinetics, hairpins with several loop and stem sequences were synthesized. Our reference system was 5′-gcUUCGgc (stem nucleotides in lower case, loop nucleotides in upper case, see Figure 1). We also studied the loop variant UUUU, as well as the stem variant 5′-ggUUCGcc. A third variant, UUC8BrG (8BrG stands for 8-bromo substituted guanine), has a G to 8BrG substitution in the loop and was previously shown to reduce the conformational entropy of the unfolded state without a large effect on the native state.15 We also made gcCGA8BrGgc and ggacCCCCgucc sequences (not discussed in detail) that served to verify that the observed multiexponential behavior could also be seen for other loop sequences or stem lengths. RNA Oligonucleotide. The unmodified 8 nt RNA oligonucleotides were purchased from Dharmacon Research. Synthesis of the 8-bromo-guanine RNA oligonucleotides was described previously.15,17,18 RNA oligonucleotides were stored in low salt buffers of P10E0.1 (10 mM sodium phosphate, 0.1 mM Na2EDTA, pH 7.1) to favor the hairpin conformation.17,19-21 (9) Chen, S. J.; Dill, K. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 646-651. (10) Ansari, A.; Kuznetsov, S. V.; Shen, Y. Q. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7771-7776. (11) Bonnet, G.; Krichevsky, O.; Libchaber, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8602-8606. (12) Ying, L. M.; Wallace, M. I.; Klenerman, D. Chem. Phys. Lett. 2001, 334, 145-150. (13) Zhang, W. B.; Chen, S. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 19311936. (14) Thirumalai, D.; Lee, N.; Woodson, S. A.; Klimov, D. K. Annu. ReV. Phys. Chem 2001, 52, 751-762. (15) Proctor, D. J.; Ma, H. R.; Kierzek, E.; Kierzek, R.; Gruebele, M.; Bevilacqua, P. C. Biochemistry 2004, 43, 14004-14014. (16) Molinaro, M.; Tinoco, I. Nucleic Acids Res. 1995, 23, 3056-3063. (17) Proctor, D. J.; Kierzek, E.; Kierzek, R.; Bevilacqua, P. C. J. Am. Chem. Soc. 2003, 125, 2390-2391. (18) Xia, T. B.; SantaLucia, J.; Burkard, M. E.; Kierzek, R.; Schroeder, S. J.; Jiao, X. Q.; Cox, C.; Turner, D. H. Biochemistry 1998, 37, 14719-14735. (19) Varani, G.; Cheong, C. J.; Tinoco, I. Biochemistry 1991, 30, 3280-3289. (20) Antao, V. P.; Tinoco, I. Nucleic Acids Res. 1992, 20, 819-824. 1524 J. AM. CHEM. SOC.
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Figure 2. Heat denaturation populations (1 ) native, 0 ) denatured) of gcUUCGgc. The UV melting curve (O) can be described as a cooperative transition with Tm ) 323 K. The four-state model calculated native population (s) is in good agreement with the experimental data.
The RNA concentration was measured by UV absorbance at 280 nm for the denatured state, prepared at 90 °C. The extinction coefficient is estimated from nearest-neighbor analysis22 and assumed to be identical for the unmodified and modified hairpins as described.17 Thermal Denaturation Before the thermodynamic measurements at pH 7, oligomers were heat denatured at 90 °C for 1-2 min. UV absorption-detected melting experiments were performed in a 1 cm path length cuvette at 260 and 280 nm, at strand concentration of 1-25 µM. Repeated melting curves are independent of the concentration within this range, indicating that duplex formation is not a major component of the melting transition as shown in Figure 2. Phosphorus NMR was used to measure the chemical shift of our reference RNA gcUUCGgc as a function of temperature. Spectra were concentration independent up to the 250 µM maximum concentration tested. Proton decoupled phosphorus NMR spectra also showed good chemical shift dispersion, seven resonances, and the unusual downfield-shifted resonance characteristic of a UUCG loop, confirming the hairpin species.15,17,24 Because the NMR data lacked complete folding and unfolding baselines, the thermodynamic parameters were determined by globally fitting the temperature dependence of the NMR and UV melting data simultaneously using a two-state nonlinear least-squares fit.15 Temperature-Jump Setup. RNA folding/unfolding kinetics were detected by jumping the temperature of RNA solutions by 7.5 °C within 10 ns. The RNA concentration range was 0.94.6 mM. Except for the CGA8BrG variant, which is not analyzed in detail here, no concentration dependence of the kinetics was detected over the ranges tested. UV absorption at 280 nm was used to detect the RNA relaxation that restores equilibrium after the temperature jump. The experiment was repeated for a range of temperatures to measure the full temperature dependence of the kinetics. The temperatures cited in the analysis are after the jump, when relaxation occurs. The laser T-jump apparatus and temperature calibration procedure have been described in detail.25-27 (21) Proctor, D. J.; Schaak, J. E.; Bevilacqua, J. M.; Falzone, C. J.; Bevilacqua, P. C. Biochemistry 2002, 41, 12062-12075. (22) Borer, P. N. Handbook of Biochemistry and Molecular Biology: Nucleic Acids, 3rd ed.; CRC Press: Cleveland, OH, 1975; Vol. I. (23) Humhrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33-38. (24) Williams, D. J.; Hall, K. B. J. Mol. Biol. 2000, 297, 1045-1061.
Energy Landscape of a Small RNA Hairpin
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The detection method (UV absorbance) differs from our previous fluorescence detection, and will be summarized briefly. A cylindrical quartz cell (Hellma) with a 100 µm path length was used to reduce distortion of the UV beam profile by the temperature jump, allowing for absorbance detection. Either no absorbance changes, or small
