Single-molecule transition-state analysis of RNA folding

Abstract

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 ...

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

“…6B). In support of this model, the formation of a compact intermediate that possesses native tertiary interactions (i.e., correctly oriented helices) is a key step during folding of large RNAs such as ribozymes (Bokinsky et al 2003;Buchmueller and Weeks 2003;Pyle et al 2007;Behrouzi et al 2012). The role of L3 very early in 60S subunit assembly is consistent with this compaction.…”

Entropy-Driven Folding of an RNA Helical Junction: An Isothermal Titration Calorimetric Analysis of the Hammerhead Ribozyme

“…However, NLPB approaches require high-resolution structural data, consider only electrostatic effects, and treat the structure of the RNA as if it were static, as opposed to the dynamic ensemble of conformations that is probably more typical for RNAs in solution. NLPB treatments are therefore most powerful as interpretive aids paired with data from other techniques, as demonstrated in recent studies employing fluorescently labeled helical junctions along with NLPB calculations to investigate junction folding and dynamics (13,16).…”

“…However, NLPB approaches require high-resolution structural data, consider only electrostatic effects, and treat the structure of the RNA as if it were static, as opposed to the dynamic ensemble of conformations that is probably more typical for RNAs in solution. NLPB treatments are therefore most powerful as interpretive aids paired with data from other techniques, as demonstrated in recent studies employing fluorescently labeled helical junctions along with NLPB calculations to investigate junction folding and dynamics (13,16).In this study, we use ITC (17) to determine thermodynamic parameters for the folding of the hammerhead ribozyme (18), a three-way RNA helical junction. Recently, Lilley and coworkers reported ITC-measured values for Mg 2+ -binding and coupled folding of the hammerhead ribozyme and two folding mutants (19).…”

Dissecting metal ion–dependent folding and catalysis of a single DNAzyme

“…FRET at the single molecule level or smFRET is a powerful method to probe dynamic conformational changes and molecular level interactions of biomolecules 29,30 . For example, smFRET studies of the hairpin ribozyme have revealed heterogeneous folding kinetics and have dissected the cleavage and ligation reactions coupled with folding and unfolding reactions 28,[31][32][33] . Here we applied smFRET to the study of the conformational changes and cleavage reactions of a DNAzyme.…”