Abstract
Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg(2+) concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding.
Highlights
Group I introns are valuable for studying RNA folding and chaperone proteins
Development of a Catalytic Activity Assay for Folding—To use catalytic activity to follow Azoarcus ribozyme folding quantitatively, it was necessary to determine the catalytic properties of the prefolded ribozyme [18]
Acceleration of Refolding by DEAD Box Proteins—We investigated the effects of the DEAD box proteins CYT-19 and Mss116p on Azoarcus ribozyme refolding
Summary
Group I introns are valuable for studying RNA folding and chaperone proteins. Results: A catalytic activity assay was developed and used to demonstrate two prominent phases for Azoarcus ribozyme folding. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Catalytic activity was used as a probe for native structure formation and revealed that, under commonly used conditions, most of the ribozyme population misfolds on this time scale to a conformation that includes extensive native structure and is not distinguished by physical probes (16 –18) This misfolded RNA refolds to the native state more slowly still, in several hours under standard conditions, in a process that involves extensive disruption of native structure [17, 19]. This RNA and its misfolded conformation should be useful in efforts to understand how RNAs misfold and how DEAD box proteins function as RNA chaperones
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