Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme (original) (raw)
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Single Molecule FRET Characterization of Large Ribozyme Folding
Methods in Molecular Biology, 2012
A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg2+ and Ca2+) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments.
Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis
Nucleic Acids Research, 2007
Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg 2þ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg 2þ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg 2þ concentration that arises from association with several Mg 2þ ions. This transition is followed by a second Mg 2þ -dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the $100 ms timescale offers insight into the folding dynamics of this ribozyme.
A Single-Molecule Study of RNA Catalysis and Folding
Science, 2000
Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules . The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.
Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study
The EMBO journal, 1997
The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a si...
The rate-limiting step in the folding of a large ribozyme without kinetic traps
Proceedings of the National Academy of Sciences, 2002
A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving preboun...
Correlating Structural Dynamics and Function in Single Ribozyme Molecules
Science, 2002
We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.
Embo Journal, 1998
The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min -1 ) but not for cleavage, where docking (0.5 min -1 ) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G ϩ1 A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.
Single VS Ribozyme Molecules Reveal Dynamic and Hierarchical Folding Toward Catalysis
Journal of Molecular Biology, 2008
Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two threeway junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.
Exploring RNA folding one molecule at a time
Current Opinion in Chemical Biology, 2008
RNA molecules fold into stable native structures to perform their biological function. RNA folding can be influenced by ions, co-factors, and proteins through numerous mechanisms. Understanding these mechanisms at the molecular level is important for elucidating the structure-function relationship in biologically important RNAs. Recent developments in single molecule spectroscopy have provided new approaches to investigate RNA folding and have allowed identification of kinetic intermediates that would otherwise remain hidden in ensemble-averaged experiments. Here we summarize some of these developments, which provide new insight into the effect of Mg 2+ ions in RNA folding landscapes, the role of cooperativity in RNA tertiary folding, the stepwise folding of RNA during transcription, and the hierarchical assembly of RNA-protein complexes.