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The hairpin ribozyme is a small catalytic motif found in plant satellite RNAs where it catalyzes a reversible self-cleavage reaction during processing of replication intermediates. Crystallographic studies of hairpin ribozymes have provided high resolution views of the RNA functional groups that comprise the active site and stimulated biochemical studies that probed the contributions of nucleobase functional groups to catalytic chemistry. The dramatic loss of activity that results from perturbation of active site architecture points to the importance of positioning and orientation in catalytic rate acceleration. The current study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the S N 2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry. Nucleotide modifications that alter or eliminate individual hydrogen bonding partners had different effects on the activation barrier to catalysis, the stability of ribozyme complexes in the ground state, and the internal equilibrium between cleavage and ligation of bound products. Furthermore, substitution of hydrogen bond donors and acceptors with seemingly equivalent pairs sometimes had very different functional consequences. These biochemical analyses augment high resolution structural information to provide insights into the functional significance of active site architecture.The well-characterized structure of the hairpin ribozyme offers a valuable framework for investigating the contributions of individual active site interactions to the activation barrier to catalysis and to the stability of ribozyme complexes in the ground state. The hairpin ribozyme catalyzes a reversible selfcleavage reaction in which nucleophilic attack of a ribose 2Ј-hydroxyl on an adjacent phosphorus proceeds through a trigonal bipyramidal transition state that leads to the formation of 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini (1). High resolution crystal structures have been solved for hairpin ribozymes in complexes with a noncleavable substrate analog, cleavage products and a vanadate mimic of the trigonal bipyramidal transition state, making this ribozyme the subject of more detailed structural analyses than virtually any other catalytic RNA (2-6) (Fig. 1).Hairpin ribozymes have two essential helix-loop-helix domains, A and B, that associate to form the active site (7). High resolution structures reveal a network of stacking and hydrogen-bonding interactions within the active site that align the reactive phosphate in the appropriate orientation for an S N 2-type nucleophilic attack mechanism and orient nucleotide base functional groups near the reactive phosphate to facilitate catalytic chemistry (Fig. 1). Gϩ1 is the conserved nucleotide on the 3Ј-side of the reactive phosphodiester in loop A (Fig. 2). Tertiary interactions between Gϩ1 and nucleotides in loop B define the architecture of the active site (2,8). Guanine at the ϩ1 posit...

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