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| The EMBO Journal
(2001) 20, 5461–5469, doi:10.1093/emboj/20.19.5461 |
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| Rapid formation of a solvent-inaccessible core in the Neurospora Varkud satellite ribozyme |
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| Shawna L. Hiley and Richard A. Collins |
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Department of Molecular and Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 To whom correspondence should be addressed Richard A. Collins, rick.collins@utoronto.ca Received 6 July 2001; Revised 1 August 2001; Accepted 13 August 2001. |
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| Abstract |
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| We have used hydroxyl radicals generated by decomposition of peroxynitrous acid to study Mg2+-dependent structure and folding of the Varkud satellite (VS) ribozyme. Protection from radical cleavage shows the existence of a solvent-inaccessible core, which includes nucleotides near two three-helix junctions, the kissing interaction between stem–loops I and V and other nucleotides, most of which have also been implicated as important for folding or activity. Kinetic folding experiments showed that the ribozyme folds very quickly, with the observed protections completely formed within 2 s of addition of MgCl2. In mutants that disrupt the kissing interaction or entirely remove stem–loop I, which contains the cleavage site, nucleotides in the three-helix junctions and a subset of those elsewhere remain protected. Unlike smaller ribozymes, the VS ribozyme retains a significant amount of structure in the absence of its substrate. Protections that depend on proper interaction between the substrate and the rest ribozyme map to a region previously proposed as the active site of the ribozyme and along both sides of helix II, identifying candidate sites of docking for the substrate helix. |
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| Keywords: hydroxyl radical footprinting, RNA folding, RNA structure, T7 RNA polymerase, VS RNA |
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