SummaryOsmolytes are small organic molecules accumulated by cells in response to osmotic stress. Although their effects on protein stability have been studied, there has been no systematic documentation of their influence on RNA. In this work, the effects of nine osmolytes on the secondary and tertiary structure stabilities of six RNA structures of differing complexity and stability have been surveyed. Using thermal melting analysis, m-values (change in ΔG° of RNA folding per molal concentration of osmolyte) have been measured. All the osmolytes destabilize RNA secondary structure, although to different extents, probably because they favor solubilization of base surfaces. Osmolyte effects on tertiary structure, however, can be either stabilizing or destabilizing. We hypothesize that the stabilizing osmolytes have unfavorable interactions with the RNA backbone, which becomes less accessible to solvent in most tertiary structures. Finally, it was found that as a larger fraction of the negative charge of an RNA tertiary structure is neutralized by hydrated Mg 2+ , the RNA becomes less responsive to stabilizing osmolytes and may even be destabilized. The natural selection of osmolytes as protective agents must have been influenced by their effects on the stabilities of functional RNA structures, though in general, the effects of osmolytes on RNA and protein stabilities do not parallel each other. Our results also suggest that some osmolytes can be useful tools for studying intrinsically unstable RNA folds and assessing the mechanisms of Mg 2+ -induced RNA stabilization.

contributed equally to this workThe catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate±pH pro®les and¯uores-cence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modi®cation of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2¢-OH and protonation of the 5¢-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto±enol tautomerization.

Many RNA tertiary structures are stable in the presence of monovalent ions alone. To evaluate the degree to which ions at or near the surfaces of such RNAs contribute to stability, the salt-dependent stabilities of a variety of RNA structures were measured with each of the five group I cations. The stabilities of hairpin secondary structures and a pseudoknot tertiary structure are insensitive to the ion identity, but the tertiary structures of two other RNAs, an adenine riboswitch and a kissing loop complex, become more stable by 2-3 kcal/mol as ion size decreases. This "default" trend is attributed to the ability of smaller ions to approach the RNA surface more closely. The degree of cation accumulation around the kissing loop complex was also inversely proportional to ion radius, perhaps because of the presence of sterically restricted pockets that can be accessed only by smaller ions. An RNA containing the tetraloop-receptor motif shows a strong (up to ∼3 kcal/mol) preference for Na + or K + over other group I ions, consistent with the chelation of K + by this motif in some crystal structures. This RNA reverts to the "default" dependence on ion size when a base forming part of the chelation site is mutated. Lastly, an RNA aptamer for cobinamide, which was originally selected in the presence of high LiCl concentrations, binds ligand more strongly in the presence of Li + than other monovalent ions.Based on these trends in RNA stability with group I ion size, it is argued that two features of RNA tertiary structures may promote strong interactions with ions at or near the RNA surface: negative charge densities that are higher than found in secondary structures, and the occasional presence of chelation sites, electronegative pockets that selectively bind ions of an optimum size.

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The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m-values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ~0 for a hairpin secondary structure and between 0.70 and 1.85 kcal/mol/m for four RNA tertiary structures (30 – 86 nts). Further analysis of two RNAs by small angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg2+, and that the structures stabilized by TMAO and Mg2+ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg2+ ion chelation site formed in part by a buried phosphate, even though Mg2+ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy +211 cal/mol/m for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66 – 173 water molecules in the RNA structures studied), and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg2+ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.