How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our singlemolecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions. RNA is the key enzymatic component in a number of essential cellular processes, such as translation and splicing (1-4). Aside from these fundamental roles, RNA also finds important applications in modern biotechnology and medicine (5, 6). For example, recent developments in small interfering RNAs, protein-binding RNA aptamers, and target-specific catalytic RNAs suggest that these functional RNAs can serve as effective tools in functional genomics and proteomics and in gene therapy (5,6). This increasing appreciation of RNA as a crucial biopolymer demands more than ever a clear picture of how RNA molecules fold into their native structures, which are vital to their functional properties. A fundamental understanding of RNA folding relies critically on the characterization of the associated folding transition states, i.e., the highest energy states along the reaction coordinates that dictate the transition kinetics. However, the characterization of the transition states of RNA folding lags far behind that of protein folding (7-11), in part because of a more rugged energy landscape for RNA that leads to multiple folding pathways and intermediate states (12)(13)(14)(15)(16)(17)(18)(19), making it difficult to characterize elementary RNA folding transitions. Here, we demonstrate a solution to this problem by using single-molecule fluorescence spectroscopy (20, 21) on a model RNA enzyme, the hairpin ribozyme.Our single-molecule time trajectories unambiguously identify multiple conformational states of the RNA and distinct structural transitions between effectively noninterchanging sets of unfolded and folded states. Using this technique, in conjunction with ...

Divalent cations, like magnesium, are crucial for the structural integrity and biological activity of RNA. In this article, we present a picture of how magnesium stabilizes a particular folded form of RNA. The overall stabilization of RNA by Mg2+ is given by the free energy of transferring RNA from a reference univalent salt solution to a mixed salt solution. This term has favorable energetic contributions from two distinct modes of binding: diffuse binding and site binding. In diffuse binding, fully hydrated Mg ions interact with the RNA via nonspecific long‐range electrostatic interactions. In site binding, dehydrated Mg2+ interacts with anionic ligands specifically arranged by the RNA fold to act as coordinating ligands for the metal ion. Each of these modes has a strong coulombic contribution to binding; however, site binding is also characterized by substantial changes in ion solvation and other nonelectrostatic contributions. We will show how these energetic differences can be exploited to experimentally distinguish between these two classes of ions using analyses of binding polynomials. We survey a number of specific systems in which Mg2+–RNA interactions have been studied. In well‐characterized systems such as certain tRNAs and some rRNA fragments these studies show that site‐bound ions can play an important role in RNA stability. However, the crucial role of diffusely bound ions is also evident. We emphasize that diffuse binding can only be described rigorously by a model that accounts for long‐range electrostatic forces. To fully understand the role of magnesium ions in RNA stability, theoretical models describing electrostatic forces in systems with complicated structures must be developed. © 1999 John Wiley & Sons, Inc. Biopoly 48: 113–135, 1998

The aim of this study is to obtain a comprehensive experimental and theoretical description of the contributions of Mg2+ ions to the free energy of folding a pseudoknot RNA tertiary structure. A fluorescence method for measuring the effective concentration of Mg2+ in the presence of an RNA was used to study Mg2+-RNA interactions with both folded and partially unfolded forms of an RNA pseudoknot. These data established the excess number of Mg2+ ions accumulated by the folded or partially unfolded RNAs as a function of bulk Mg2+ concentration, from which free energies of Mg2+-RNA interactions were derived. Complementary thermal melting experiments were also used to obtain RNA-folding free energies. These experimental data were compared with the results of calculations based on the nonlinear Poisson-Boltzmann equation, which describes the interaction of "diffuse" (fully hydrated) Mg2+ ions with the different RNA forms. Good agreement between the calculations and experimental data suggests that essentially all of the Mg2+-induced stabilization of the native pseudoknot structure arises from the stronger interaction of diffuse ions with the folded tertiary structure compared to that with a partially unfolded state. It is unlikely that the stability of the RNA depends on dehydrated ions bound to specific sets of RNA ligands in the folded state. The data also suggest that the Mg2+-dependent free energy of folding is sensitive to factors that influence the ensemble of RNA conformations present in the partially unfolded state.