Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

Abstract

Single-molecule fluorescence resonance energy transfer studies of freely diffusing hairpin ribozymes with different combinations of helical junction and loop elements reveal striking differences in their folding behavior. We examined a series of six different ribozymes consisting of two-, three-and four-way junction variants, as well as corresponding constructs with one of the two loops removed. Our results highlight the varying contributions of preformed secondary structure elements to tertiary folding of the hairpin ribozyme. Of the three helical junction variants studied, the four-way junction strongly favored folding to a docked conformation of the two loops, required for catalytic activity. Moreover, the four-way junction was uniquely able to fold to a similar compact structure even in the absence of specific loop-loop docking interactions. A key feature of the data is the observation of broadening/tailing in the fluorescence resonance energy transfer histogram peak for a single-loop mutant of the four-way junction at higher Mg 21 concentrations, not observed for any of the other single-loop variants. This feature is consistent with interconversion between compact and extended structures, which we estimate takes place on the 100ms timescale using a simple model for the peak shape. This unique ability of the four-way junction ribozyme to populate an undocked conformation with native-like structure (a quasi-docked state) likely contributes to its greater tertiary structure stability, with the quasi-docked state acting as an intermediate and facilitating the subsequent formation of the specific hydrogen bonding network during docking of the two loops. The inability of two-and three-way junction ribozymes to fully populate a docked conformation reveals the importance of correct helical junction geometry as well as loop elements for effective ribozyme folding.

Single-Molecule Fluorescence Resonance Energy Transfer Assays Reveal Heterogeneous Folding Ensembles in a Simple RNA Stem–Loop

“…This has led to a number of SM studies mostly directed at understanding the molecular mechanism of RNA catalysis and the folding of small ribozymes into functional states. [30][31][32] RNA stem-loop formation has previously been probed by temperaturejump experiments, and, more recently, the folding and dynamics of such molecules have been the subject of a number of theoretical and experimental articles. 33 Since stem-loop formation is the first step in creating higher-order RNA folds 34 that often serve as critical recognition sites for other ligands that can be both temporally and spatially regulated, it is important that our understanding encompasses the behaviour of these RNA structural elements.…”

Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization

“…Fluorescence emission from one or several fluorophores can be used to establish the absolute position of the single molecules with application for DNA (or RNA) sequence mapping (Gordon et al 2004;Qu et al 2004). Fluorescence correlation spectroscopy (FCS) has been employed to establish the diffusion constant and hydrodynamic radius to establish the (folded) size of RNA (Bonnet et al 1998;Pljevaljcic et al 2004). In all cases, site-specific attachment of fluorophores with specific fluorescence or environment-sensitive properties is a requirement.…”

Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting”

“…7]. The reaction mechanism of the hairpin ribozyme has been explored by ensemble kinetic and structural studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and singlemolecule experiments (16)(17)(18)(19)(20)(21). The enzyme catalyzes the cleavage reaction in several steps (Fig.…”