Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif.
The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.
Klostermeier
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The Scripps Research Institute, Department of Molecular Biology, MB-19, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Millar
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D004735:Energy Transfer; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008962:Models, Theoretical; D017860:Nepovirus; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D016337:RNA, Catalytic / Q000737:chemistry*; D019244:RNA, Satellite / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry; D013816:Thermodynamics
D016337:RNA, Catalytic; D019244:RNA, Satellite; D012367:RNA, Viral; C418481:hairpin ribozyme; D012266:Ribose