The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

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.

mRNA secondary structures fold sequentially but exchange rapidly in vivo.

RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme.

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions 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 in order 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.

Functional analysis of hairpin ribozyme active site architecture.

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.

Role of an active site adenine in hairpin ribozyme catalysis.

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.