The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5'-GU-3' immediately 5' of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and 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 common interdomain distance as measured by time-resolved fluorescence resonance 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 kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.