Functional Analysis of Hairpin Ribozyme Active Site Architecture

Abstract

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...

Electrostatic interactions in the hairpin ribozyme account for the majority of the rate acceleration without chemical participation by nucleobases

“…These results are in accord with the experimental estimate of 20-21 kcal/mol (Nesbitt et al 1999;Fedor 2000;Kuzmin et al 2005). Nonetheless, this does not preclude alternate mechanisms with explicit nucleobase involvement, such as A38 and G8 acting as general acid and general base catalysts (Rupert et al 2002;Ferré-D'Amaré 2004;Cottrell et al 2007), respectively, which could further lower the barrier. In order to address the question as to what extent the electrostatic environment and nonspecific interactions alone can account for the observed rate enhancement, electrostatic solvation free energies are computed at various stationary points on the free energy surface and are compared to the values at the reactant state.…”

“…The hairpin ribozyme (HPR) (Walter and Burke 1998;Lilley 1999), which catalyzes the reversible, site-specific phosphodiester bond cleavage of an RNA substrate, is unique in that the chemical steps of the reaction do not require a specific metal ion in the active site (Walter and Burke 1998;Lilley 1999;Doherty and Doudna 2001;Rupert et al 2002;Bevilacqua 2003) but is able to achieve rate acceleration similar to other metal-dependent ribozymes (Fedor 2000). There is an active debate regarding the origins of the catalytic proficiency of HPR, focusing mostly on the role of active site nucleobases in general acid and base catalysis (Hampel and Cowan 1997;Nesbitt et al 1999;Fedor 2000;Pinard et al 2001;Ryder and Strobel 2002;Kuzmin et al 2005;Cottrell et al 2007;Tang et al 2007). X-ray crystallographic structures of HPR provide valuable insight into the active site conformation in the reactant state (Rupert and Ferré-D'Amaré 2001;Salter et al 2006), transition state, and product state mimic structures (Rupert et al 2002;Torelli et al 2007).…”

“…3 In this report, we show that the electrostatic environment provided by solvated HPR active site lowers the cleavage activation barrier up to 9 kcal/mol relative to the uncatalyzed transphosphorylation barrier in water. We also explore the mechanistic scenarios whereby G8 acts as a general base to activate the 29-hydroxyl nucleophile, as implicated by experiment (Pinard et al 2001;Rupert et al 2002;Wilson et al 2006;Cottrell et al 2007). The results suggest that the electrostatic environment of the solvated ribozyme active site contributes significantly in achieving 10 6 -10 7 -fold rate enhancement of the phosphodiester cleavage (Nesbitt et al 1999;Fedor 2000;Kuzmin et al 2005) relative to the uncatalyzed, but spontaneous, cleavage of RNA molecule in aqueous solution (Hertel et al 1997;Li and Breaker 1999).…”

Quantum Mechanical/Molecular Mechanical Simulation Study of the Mechanism of Hairpin Ribozyme Catalysis

“…On the basis of the transition state mimic structure with a pentavalent vanadate ion at the scissile phosphate position, it has been suggested that A38, in protonated form, acts as a general acid in the cleavage reaction. 4,24,25 The G8 nucleotide base has been implicated in the stabilization of the transition state. 4,25 This is consistent with the results from nucleotide analog interference mapping experiments.…”

“…26,27 G8 and A38 are critical in catalysis: the deletion of the G8 base decreases the reaction rate by a factor of 350, and the deletion of the A38 base decreases the rate more than 10 000-fold relative to the wild-type control. 9,24,28,29 One important factor is the protonation states of the scissile phosphate and other nucleotides present in the active site and their relationship to the pH-rate profiles. Although G8, A38, and other nucleobases have been found to significantly affect the catalytic efficiency, 26,27,30,31 their specific roles in catalysis have not yet been clearly defined.…”