The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in posttranscriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.Hfq protein from Escherichia coli was first described in connection with Qβ-phage replication 1,2 . Hfq has recently emerged as a central player in post-transcriptional gene regulation as mediated by bacterial ncRNAs [3][4][5][6] . Escherichia coli Hfq mutants show disrupted signaling in stress response pathways 7,8 , arising from the need for Hfq to mediate base-pairing between regulatory ncRNAs and their mRNA targets. Examples of these partnerships include DsrA-rpoS 7,9,10 , OxyS-fhlA 11,12 , OxyS-rpoS 13 , RprA-rpoS 14 , RyhBsodB [15][16][17] .Complexes between ncRNAs and their mRNA targets function in several ways. Most commonly, complexed structures lead to translational activation or repression by remodeling mRNA regulatory regions containing the ribosome-binding site (RBS) and/or start codon. Alternatively, the interaction can enhance decay of the target mRNA16 or simply block translation11. Clearly, Hfq facilitates base-pairing between ncRNAs and their targets, but how it does so is poorly understood. How the chaperone function relates to other Hfq activities such as the control of poly(A) tail elongation19 , 20 and regulation of mRNA stability21 , 22 is also unknown. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2011 April 5. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHfq shares sequence similarity to the eukaryotic Lsm proteins [23][24][25][26][27] We addressed these questions through a mutational analysis of Hfq, probing in vitro binding to several model RNAs that represent species with which Hfq must interact. Hfq mutants were assayed in vivo using a reporter assay and RNA lifetime experiments. Together, the results support a model wherein at least two independent RNA-binding sites exist on the Hfq hexamer, and juxtaposition of bound RNAs facilitates base-pairing. RESULTS Hfq mutagenesisTo identify amino acids essential for RNA binding, we constructed a series of E. coli Hfq misse...

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Whereas heat capacity changes (ΔC P s) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of ΔC P s associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of ΔC P s and points toward the challenges that will need to be overcome if the energetic contribution of ΔC P terms are to be put to use in improving free energy calculations for nucleic acid structure prediction.

Duplexes are the most fundamental elements of nucleic acid folding. Although it has become increasingly clear that duplex formation can be associated with a significant change in heat capacity (ΔC p ), this parameter is typically overlooked in thermodynamic studies of nucleic acid folding. Analogy to protein folding suggests that base stacking events coupled to duplex formation should give rise to a ΔC p due to the release of waters solvating aromatic surfaces of nucleotide bases. In previous work, we showed that the ΔC p observed by isothermal titration calorimetry (ITC) for RNA duplex formation depended on salt and sequence. In the present work, we apply calorimetric and spectroscopic techniques to a series of designed DNA duplexes to demonstrate that both the salt dependence and sequence dependence of ΔC p s observed by ITC reflect perturbations to the same fundamental phenomenon: stacking in the single-stranded state. By measuring the thermodynamics of single strand melting, one can accurately predict the ΔC p s observed for duplex formation by ITC at high and low ionic strength. We discuss our results in light of the larger issue of contributions to ΔC p from coupled equilibria and conclude that observed ΔC p s can be useful indicators of intermediate states in nucleic acid folding phenomena.Phase transitions within systems of many weak interactions are generally associated with a significant change in heat capacity (1-3). Macromolecular folding events are examples of such transitions, and heat capacity changes (ΔC p s) have been measured for folding in many proteins (summarized in ref 4) and an increasing number of nucleic acids (5). When ΔC p is large relative to ΔS for the same transition, the ΔC p can significantly perturb fold stability (ΔG) as a function of temperature (6). Large ΔC p s can result in the phenomenon of cold denaturation, as observed for proteins (7-9) and recently for RNA (10,11). Usually, ΔC p is thought to arise from solvent effects that accompany the burial of hydro-phobic and/or aromatic surfaces upon folding. In nucleic acids, residual stacking in the single-stranded state is also temperature dependent and may reduce the amount of aromatic surface exposed in the unfolded state under some conditions, thus complicating the interpretation of observed ΔC p s. While ΔC p s associated with protein folding have been investigated in great detail (e.g., ref 23 and references cited therein), there have been fewer systematic studies of the ΔC p of nucleic acid folding (6,11,12,20,21,(24)(25)(26)(27)(28)(29)(30).The origin of ΔC p s associated with protein folding has been thoroughly examined (4,23,31,32). Empirically, the ΔC p has been found to be proportional to the amount of hydrophobic surface area buried during folding (23). Methodes employing solute transfer data for small molecule model compounds (and an assumption of group additivity) can largely account for † This work was supported by a research grant from the NIH (GM-065430 to A.L.F.). A.L.F. is a Cottrell Scholar of the R...

In proteins, empirical correlations have shown that changes in heat capacity (DeltaC(P)) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(P) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaC(P)s of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(P) of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(P) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(P). We weigh the strengths and limitations of this method for determining DeltaC(P) values. Finally, we discuss the data in light of the physical origins of the DeltaC(P)s for RNA folding and consider their impact on biological function.

Cold denaturation is a phenomenon common to many proteins, 1 but it has not previously been observed directly for nucleic acids. 2 It results from a difference in the heat capacities (ΔC p ) of the folded and unfolded states, and correlates with the change in accessible surface area between the two species. 3 Incorporation of a ΔC p for (un)folding into the Gibbs free energy results in a modified form of the Gibbs equation (eqs 1 and 2), where T* is an arbitrary reference temperature, and ΔH* and ΔS* are the enthalpy and entropy, respectively, at that temperature. Large ΔC p terms result in curvature of the free energy profile such that a temperature of maximum stability exists, flanked by hot and cold melting temperatures (T m 's).(1) (2)The ΔC p for nucleic acid folding has been commonly assumed to be approximately zero. [4][5][6] However, several recent studies reported significant ΔC p 's for DNA duplex formation [7][8][9] and RNA tertiary folding, 10-13 leading to predicitions of cold unfolding. Here, we report spectroscopic evidence that the hammerhead ribozyme (Figure 1), a small self-cleaving RNA, 14 undergoes cold denaturation, the first direct observation of this phenomenon for a nucleic acid.Recent cryoenzymological studies on hammerhead ribozyme 16 (HH16) demonstrated that a 40% (v/v) methanol in water cryosolvent system only minimally perturbed the ribozyme, maintaining 80% of the rate observed in aqueous solution, with no loss in total extent of cleavage. 15 The cryosolvent allowed kinetic measurements of activity at temperatures down to −33 °C. A dramatic reduction in cleavage rate occurred below −27 °C, yielding significant curvature in the Eyring plot. Among the possible explanations for these data was a cold denaturation transition inactivating the ribozyme.The low-temperature structure of HH16 was therefore investigated, using circular dichroism (CD) spectroscopy. 16 CD is an effective probe of nucleic acid secondary and tertiary structure. 17 FRET,21 and calorimetry 11 (data not shown). At 20 °C, addition of 40% methanol to folded samples of HH16 causes no significant change in the CD spectrum. Upon cooling, however, a dramatic change occurs between −10 and −30 °C under conditions similar to those used for cryoenzymology, indicative of a near total loss of secondary and tertiary structure ( Figure 2). Under these conditions, data monitored at either 264 or 209 nm do not fit a two-state model, but qualitatively resemble two overlapping transitions possibly corresponding to sequential loss of tertiary and secondary structures. Control experiments employing radiolabeled HH16 provide no evidence of precipitation down to −30 °C, the lowest temperature at which we could assay for precipitate by centrifugation.Optimization of the pH and ion concentrations yielded conditions under which the structural transitions are reversible and fit nicely to a two-state model. 22 In 10 mM Mg 2+ at pH 5.0, a transition with a T m of −20 °C is observed (see Figure S1).The amplitude of this structural change, in ...

Helical junctions are extremely common motifs in naturally occurring RNAs, but little is known about the thermodynamics that drive their folding. Studies of junction folding face several challenges: non-two-state folding behavior, superposition of secondary and tertiary structural energetics, and drastically opposing enthalpic and entropic contributions to folding. Here we describe a thermodynamic dissection of the folding of the hammerhead ribozyme, a three-way RNA helical junction, by using isothermal titration calorimetry of bimolecular RNA constructs. By using this method, we show that tertiary folding of the hammerhead core occurs with a highly unfavorable enthalpy change, and is therefore entropically driven. Furthermore, the enthalpies and heat capacities of core folding are the same whether supported by monovalent or divalent ions. These properties appear to be general to the core sequence of bimolecular hammerhead constructs. We present a model for the ion-induced folding of the hammerhead core that is similar to those advanced for the folding of much larger RNAs, involving ion-induced collapse to a structured, non-native state accompanied by rearrangement of core residues to produce the native fold. In agreement with previous enzymological and structural studies, our thermodynamic data suggest that the hammerhead structure is stabilized in vitro predominantly by diffusely bound ions. Our approach addresses several significant challenges that accompany the study of junction folding, and should prove useful in defining the thermodynamic determinants of stability in these important RNA motifs.RNA helical junctions, single-stranded loops flanked by multiple double helices, are pervasive in known and predicted RNA structures. Helical junctions appear in a broad range of functional RNAs (1,2), including small and large ribozymes, mRNA untranslated regions, riboregulatory RNAs, snRNAs, and rRNAs. Alone or in complex with proteins, junctions may serve as the major tertiary structural elements of small RNAs or as critical elements in organizing much larger architectures. Despite their prominence, little is known about the forces that drive the folding of RNA helical junctions. Pairwise coaxial stacking and ion-dependent folding have emerged as common themes in junction architecture, but the exact role of central, looped regions and the energetics of the folding process are still unclear. As a result, there are currently no general principles of helical junction folding dependable enough for use in predicting RNA tertiary structure, much less in predicting the impact of solution conditions or bound ligands on junction stabilities. † This work was supported by IU, the IU Department of Chemistry, grants from the NIH (GM-065430 to A.L.F. and T32-GM07757 to IU/P.J.M.), the NSF (CHE-9909407 to A.L.F.) and an HHMI/Capstone award to J.C.T. Andrew Feig is a Cottrell Scholar of Research Corporation.*To whom correspondence should be addressed:Andrew L. Feig, Department of Chemistry, Indiana University, 800 E. Kirk...

RNAs were recently shown to undergo a low-temperature unfolding reaction, a phenomenon called cold denaturation. 1 Cold denaturation occurs when a macromolecule folds in such a way that it is accompanied by a large change in the heat capacity of the polymer (ΔC P ). 2-5 Typically, this ΔC P term derives from solvent effects and the burial of hydrophobic surfaces upon folding. In the case of nucleic acids, added complexity may arise from residual stacking in the single-stranded state that reduces the amount of hydrophobic surface exposed in the unfolded state. 6 While ΔC P of protein folding has been studied extensively, 7 the number of studies addressing the ΔC P of nucleic acid folding is much smaller. 6,8-12 Previous predictions for nucleic acid duplexes had estimated that this unfolding would not occur above temperatures around −120 °C. 13 When cold denaturation of RNA was discovered, the finding indicated that the ΔC P for RNA folding was significantly larger than previously expected. 14 Thus, the discovery of RNA cold denaturation prompted us to begin a detailed investigation of the role that ΔC P might play in RNA stability and the solution conditions that affect it. Herein, we report our discovery that the ΔC P for two simple RNA duplexes (Scheme 1) exhibit a marked ionic strength dependence.The nature of ΔC P effects in protein folding has been well studied. 7,15,16 The ΔC P upon folding can be measured directly through calorimetry or estimated from chaotropic unfolding studies conducted at several temperatures. Empirically, ΔC P scales with the size of the protein and the extent of hydrophobic burial during folding. 7 One explanation for this phenomenon involves the release of water molecules that form clathrate structures around the hydrophobic side chains in the unfolded state. While the magnitude of the ΔC P for protein folding is modulated by organic cosolvents, 17 this parameter is relatively insensitive to the solution ionic strength, unless specific ion binding occurs upon folding. 18 Much less is known about the solution parameters that affect ΔC P for nucleic acid folding. One might predict that ΔC P s would be modulated by the ionic strength of the solution through site-specific binding or ion condensation effects.RNA duplex formation is the most fundamental element in RNA folding. Although residual base stacking can be present in unfolded RNAs, the base pairing process generally is coupled to base stacking and thus involves occlusion of the planar hydrophobic surface. Since water is excluded as part of this process, analogy to protein folding suggests that a significant ΔC P should accompany duplex formation. In many studies of duplex thermodynamics, ΔC P effects are ignored. When they are considered, it is often as a small per base pair contribution of −20 to −200 cal mol −1 bp −1 K −1 . 6,8,12 The quantitative disparity might imply significant solution effects or nearest neighbor contributions which affect the magnitude of the ΔC P .E-mail: afeig@indiana.edu. Experimental procedures and DSC...