Monitoring of an RNA Multistep Folding Pathway by Isothermal Titration Calorimetry

Reymond, Cédric; Bisaillon, Martin; Perreault, Jean‐Pierre

doi:10.1016/j.bpj.2008.09.033

Cited by 12 publications

(26 citation statements)

References 30 publications

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“…Although differing in magnitude, each of these systems exhibits significant enthalpic gain (Δ H ° < 0) and entropic penalty (Δ S ° < 0) for folding. Furthermore, in a recent ITC study, Reymond et al demonstrated through systematic mutations that various steps in the folding pathway of the hepatitis delta virus ribozyme are exothermic and entropically disfavored (24). However, these observations of enthalpy-driven RNA folding are not a universal trend, as evidenced in Table 2.…”

Section: Discussionmentioning

confidence: 99%

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop−Receptor Tertiary Interaction

et al. 2009

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RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 °C), from which a van't Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl 2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol −1 K −1 ) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol −1 K −1 ) species. Kinetic heterogeneity in the tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop-receptor interaction can account for ~60% of the ΔH°a nd ΔS° of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy-versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.RNA folding is generally hierarchical, with tertiary structure occurring through interactions of preformed secondary elements (1-3). As a result, the kinetics and thermodynamics of tertiary interactions are crucial to understanding RNA folding and functionality as well for accurate structural predictions (1,4). Toward this end, individual folding motifs must be characterized both in isolation and in combination for a unifying thermodynamic description of RNA folding † This work was supported in part by the NSF, NIST, the W. M. Keck Foundation initiative in RNA sciences at the University of Colorado, Boulder, and PicoQuant, GmbH. J.L.F. was supported in part by Optical Science and Engineering Program NSF-IGERT and CU Biophysics Training (T32 GM-065103) grants.© 2009 American Chemical Society * To whom correspondence should be addressed: JILA, University of Colorado, 440 UCB, Boulder, CO 80309-0440. djn@jila.colorado.edu. Phone: (303) . SUPPORTING INFORMATION AVAILABLEPrediction of the effect of donor quantum yield (Q D ) on the observed FRET efficiency (E FRET ) as a function of Cy3-Cy5 distance (R) ( Figure S1) and mean cross correlations of donor and acceptor channels for the same sample containing tetraloop-receptor constructs under freely diffusing single-molecule conditions at 21 and 45 °C ( Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ...

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Section: Discussionmentioning

confidence: 99%

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop−Receptor Tertiary Interaction

et al. 2009

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“…binding to the ribozyme (Zamel and Collins 2002), it used a ribozyme system stabilized by an artificial intermolecular stem and thus yielded K D values that do not directly reflect the stability of the I/V kissing loop (Zamel and Collins 2002). In the present work, we use isothermal titration calorimetry (ITC) (Reymond et al 2009;Salim and Feig 2009) to thermodynamically characterize the I/V kissing-loop interaction formed between isolated SLI and SLV hairpins. As a result, our study provides K D values as well as energetic contributions directly associated with formation of the I/V kissing-loop interaction.…”

Section: Introductionmentioning

confidence: 99%

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

Bouchard

Legault

2014

RNA

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Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissingloop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

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“…The RNA folding pathway is typically enthalpy driven, and the formation of numerous tertiary interactions appears to be crucial in obtaining increasingly stable intermediates. However, our understanding of how these interactions take place remains scanty (Moody and Bevilacqua, 2003;Reymond et al, 2009a;Fiore et al, 2009). Due to the energies involved in the formation of RNA helices, the secondary structure is formed significantly faster than the tertiary structure (Sosnick and Pan, 2004).…”

Section: Introductionmentioning

confidence: 99%

Developing Three-Dimensional Models of Putative-Folding Intermediates of the HDV Ribozyme

et al. 2010

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Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway.

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Monitoring of an RNA Multistep Folding Pathway by Isothermal Titration Calorimetry

Cited by 12 publications

References 30 publications

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop−Receptor Tertiary Interaction

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop−Receptor Tertiary Interaction

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

Developing Three-Dimensional Models of Putative-Folding Intermediates of the HDV Ribozyme

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