title: The Azoarcus group I intron ribozyme misfolds and is accelerated for refolding by ATP-dependent RNA chaperone proteins.
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.
authors: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
authors: Selma
authors: S
authors: Sinan
authors: Xiaoyan
authors: X
authors: Yuan
authors: Rick
authors: R
authors: Russell
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D000255:Adenosine Triphosphate; D053487:DEAD-box RNA Helicases