Energetics of hydrogen bond networks in RNA: hydrogen bonds surrounding G+1 and U42 are the major determinants for the tertiary structure stability of the hairpin ribozyme.

ABSTRACT

The hairpin ribozyme, a small catalytic RNA consisting of two helix-loop-helix motifs, serves as a paradigm for RNA folding. In the active conformer, the ribozyme is docked into a compact structure via loop-loop interactions. The crystal structure of the docked hairpin ribozyme shows an intricate network of hydrogen bonding interactions at the docking interface, mediated by the base, sugar, and phosphate groups of U42 and G+1 [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786]. To elucidate the determinants for tertiary structure stability in the hairpin ribozyme, we evaluated the energetic contributions of hydrogen bonds surrounding U42 and G+1 by time-resolved fluorescence resonance energy transfer using modified ribozymes that lack one or more of the individual interactions. Elimination of a single tertiary hydrogen bond consistently resulted in a net destabilization of approximately 2 kJ/mol. The results of double- and triple-mutant cycles suggest that individual hydrogen bonds surrounding G+1 or U42 act cooperatively and form extended hydrogen bond networks that stabilize the docked ribozyme. These results demonstrate that RNAs, similar to proteins, can exploit coupled hydrogen bond networks to stabilize the docking of distant structural domains.