No abstract

Self-cleavage assays of RNA folding reveal that mRNA structures fold sequentially in vitro and in vivo, but exchange between adjacent structures is much faster in vivo than it is in vitro.

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise function(s) in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8 and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK a values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK a for ionization of 8azaA at position 38 with the apparent pK a for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK a values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5-oxygen-phosphorus bond.Hairpin ribozymes (Hp Rz) 2 belong to one of several families of small self-cleaving RNAs that serve as useful models of RNA catalysis because they are relatively simple and amenable to chemogenetic analyses (1). The Hp Rz remains functional in the absence of divalent metals, relying exclusively on nucleotide functional groups for catalytic chemistry (2-5). High resolution structures of the Hp Rz bound to transition state mimics show two active site purines, G8 and A38, positioned in a manner similar to the two histidines in RNase A that mediate general acid-base catalysis of the same reaction ( Fig. 1A) (6 -8), leading to the proposal that the Hp Rz uses the same concerted general acid-base mechanism (Fig. 1B) (9). A38(N1) is near the 5Ј-oxygen of the reactive phosphodiester, and A38(N6) lies within hydrogen bonding distance of the pro-R P non-bridging oxygen. Exogenous nucleobase rescue experiments confirmed that the amidine group of A38 interacts with the transition state, but its precise role remained unclear (10). In a general acid-base model, the cationic protonated form of A38 would act as a general acid during cleavage to donate a proton to the 5Ј-oxygen leaving group as the 5Ј-oxygen-phosphorus bond breaks. During the reverse ligation reaction, unprotonated A38 would accept a proton to activate the nucleophilic 5Ј-oxygen for attack on phosphorus as the 5Ј-oxygen-phosphorus bond forms. Cleavage and ligation rate constants both increase with increasing pH and exhibit the same apparent pK a values near 6.5 (3), consistent with the law of microscopic reversibility (11). The pH dependence of reaction kinetics can provide information about the identity of general acid-base catalyst when an apparent pK a value clearly corresponds to the microscopic pK a value for a particular active site functional group. However, no RNA functional groups exhibit protonation equilibria near pH 6.5, at least as free nucleotides in solu...

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...

The hairpin ribozyme mediates catalysis through nucleotide functional groups, without metal cation cofactors. The positions of G8 and A38 in the ribozyme active site resemble the orientation of two histidines in the active site of ribonuclease A, leading to the model that G8 and A38 mediate catalysis through a similar general acid base mechanism. However, adenosine and guanosine undergo ionization only at pH extremes, at least in solution, which seems to make them poor acid base catalysts. We used 8‐azapurine fluorescence to learn whether purine ionization equilibria change in the active site relative to pKa values in solution. 8‐azapurines display high fluorescence emission intensity when N1 is deprotonated and low intensity when N1 is protonated. A ribozyme with 8azaG8 exhibits full catalytic activity and cleaves with an apparent pKa value in the neutral range, similar to an unmodified ribozyme. Microscopic pKa values for deprotonation of 8azaG in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self‐cleavage kinetics. Thus, the increase in self‐cleavage activity with increasing pH does not reflect G8 deprotonation and G8 is mostly protonated at neutral pH. A simple interpretation of these results is that G8 functions in the protonated form, perhaps by donating hydrogen bonds that stabilize the transition state.