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45534-AC4
Water as a Specific Acid/Base Catalyst in the Mechanism of Action of a Non-Coding Catalytic RNA
Ribozymes are naturally occurring RNA enzymes that catalyze a diverse number of biological reactions such as protein synthesis, intron splicing and tRNA processing. The preponderance of evidence suggests that the chemical strategies employed by ribozymes to accelerate reaction rates are comparable, but less efficient, than those of protein enzymes. To elucidate the principles of ribozyme catalysis, the Wedekind lab studies the hairpin ribozyme, a small non-coding RNA from satellite tobacco ringspot virus. During the course of recent crystallographic investigations on this subviral plant pathogen, ordered water molecules were detected within a solvent sequestered pocket located in the active site near nucleobases A9 and A10. Due to the limited functional group repertoire of RNA enzymes, it has been hypothesized that such water molecules may participate directly in the reaction, possibly by serving as specific acid or base catalysts, or in a proton relay accessible only in the transition state.
The goal of this research is to identify and assess the role of key waters in the hairpin ribozyme active site. The significance of such waters will be evaluated initially by their locations in x-ray crystal structures representative of pre-catalytic, reaction intermediate and product-bound conformational states (Aim 1). Subsequently, a series of modified purine nucleotides will be synthesized and incorporated into the hairpin ribozyme active site to displace specific waters. Such modified ribozymes will be subjected to single-turnover activity assays and analyzed crystallographically for the ability to adopt near-native structural folds (Aim 2).
Significant progress has been made on both aims during the past year. First, we improved the refinement of the pre-catalytic structure of the hairpin ribozyme, and developed a sensitive single-turnover assay that demonstrates fast-phase cleavage kinetics (~0.4 min-1) for minimal, hinged ‘wild-type' crystallization constructs. Next, we solved high resolution crystal structures of hinged hairpin ribozymes in complex with vanadium and a novel 2'-5'-linkage; these two structures represent transition-state and reaction-intermediate analogs, respectively. Notably, the resolution of the vanadate complex was extended to 2.05 Å resolution, whereas that of the 2'-5' linkage was 2.3 Å resolution. Both structures provided evidence for the participation of key waters (W5 & W7) in transition-state stabilization, which complements the electrostatic stabilization afforded by the exocyclic amines of G8, A9 and A38. In terms of the product complex, synthesis of the N1-deazaadenosine nucleoside was completed, and so was the generation of 5'-deoxy-5'-fluoroguanosine. Both nucleosides are being prepared as phosphoramidites for RNA chemical synthesis. It is anticipated that the structures of these product mimic complexes will be solved during the next year. A product mimic structure devoid of the 2',3'-cyclic phosphate was solved during the past year as a basis for comparison to the vanadate and 2'-5' complexes.
As hypothesized, conserved waters (W5 & W7) have been identified in the A9/A10 pocket of all crystal structures that are representative of reaction intermediates (Aim 1). As such, synthesis of an N1-alkyladenosine library has been undertaken with the goal of blocking the binding of waters within this pocket by selective placement of bulky hydrocarbon substituents at the N1 atom of A9 or A10. Notably, the synthetic strategy proposed originally, which started from 2,3-diamino-5-bromopyridine, has been altered in favor of a more direct route to products. This change expands upon the work of Herdewijn & co-workers who synthesized 1-methyl adenosine for incorporation into tRNA. Thus far, the production of 3 alkylated adenosine nucleosides (methyl, ethyl and propyl) has been achieved. The next step involves conversion of each library member into a phosphoramidite for RNA chemical synthesis. Future plans include the addition of alcohol substituents onto the alkyl moiety, which was proposed to serve as a hydrogen bond donor/acceptor functionality. The enzymatic activity and structures of library members will be assessed during the next year.
Perhaps the most important discovery associated with this grant was the finding that water molecules appear to stabilize the hairpin ribozyme transition state. This concept is reasonable since nucleic acids do not possess the highly polarized amide backbone and positively-charged side chains observed in proteins. As such, it is difficult to evolve a densely packed pocket within an RNA fold that counters localized negative charge associated with a transition state. Hence, the ability to selectively coordinate water may be a general feature of RNA enzymes. This hypothesis will be tested directly for the hairpin ribozyme in the coming year.
Overall, this work has positively influenced the way the scientific community perceives RNA enzymes with respect to the role of water. The results, which have been deposited as atomic coordinates into the public PDB database, have served as the basis for several new ribozyme investigations. The ability of this work to impact the field in such a short period is a credit to the PRF and its sponsors, who are greatly acknowledged for their support.
