A base change in the catalytic core of the hairpin ribozyme perturbs function but not domain docking

The hairpin ribozyme is a small endonucleolytic RNA motif with potential fo r targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5'-GU-3' immediately 5' of G. Previously, we have shown that tert iary structure docking of its two domains is an essential step in the react ion pathway of the hairpin ribozyme. Here we show, combining biochemical an d fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a co mmon interdomain distance as measured by time-resolved fluorescence resonan ce energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetical ly more complex, yet with an equilibrium shifted toward the docked conforma tion. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is obser ved. This substrate straining accompanies global domain docking and involve s unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These d ata appear to invoke a mechanism of functional interference by a single bas e mutation, in which the ribozyme-substrate complex becomes trapped in a na tivelike fold preceding the chemical transition state.