This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The hairpin ribozyme is small catalytic RNA motif that does not utilize metal ions directly in bond scission. This suggests a mechanism of action involving: (i) transition-state stabilization, (ii) general acid/base catalysis or (iii) specific acid/base catalysis, possibly through activated water(s). The applicant?s lab has chemically synthesized and crystallized a minimal hairpin ribozyme in the presence of several nucleotide-variants. A38C, A38isoG, A38AP (i.e. aminopurine) resulted in loss-of-function, whereas A38DAP (di-AP) resulted in a gain-of-function in activity assays. Crystals of these variants have been prepared in forms presenting the ?pre-catalytic? and ?transition-state? geometries. Previously, the minimal hairpin ribozyme structure was determined at 2 resolution by the applicant's lab. The diffraction data resulting from this proposal will identify structural features (such as ordered waters and interaction distances) that have been invoked to support one mechanism of action over another.
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