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 consist s of two helix-loop-helix elements on two adjacent arms of a four-way helic al junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prere quisite for catalysis. The two loops interact via a ribose zipper motif inv olving the 2-hydroxyls of A(10), G(11), A(24), and C-25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energeti c importance of the ribose zipper hydrogen bonds, we have incorporated deox y modifications at these four positions and determined the resulting destab ilization of the docked conformer by means of time-resolved fluorescence re sonance energy transfer. In a minimal form of the ribozyme, in which the lo ops 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 n atural four-way junction ribozyme, suggesting that hydrogen bonding interac tions involving the 2 ' -hydroxyls do not contribute to the stability of th e docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ sig nificantly for the minimal and natural forms of the ribozyme.