This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have obtained a set of crystallographic snapshots of RNA catalysis using crystals of an active hammerhead ribozyme in conjunction with chemical and physical trapping techniques that have enabled us to conduct time-resolved monochromatic x-ray crystallographic analyses of the ribozyme's reaction coordinate. To date, we have obtained structures of the initial state of the ribozyme, three pre-catalytic presumed intermediate conformations, and a preliminary structure of the cleaved form of the RNA. Together, these crystal structures have allowed us to construct a simple 'movie' depicting the structural changes that appear to accompany catalysis. The third pre-catalytic intermediate fortuitously revealed that interactions between two helical stems regulate cleavage activity. This observation, as well as publication of a longer, wild-type sequence in which extensive Stem I - Stem II interactions are observed to greatly accelerate either the forward or the reverse reaction rates (separately), compel us to try to understand the structural basis of this nuclease-ligase switching. The switch between nuclease and ligase activity is required in the life cycles of RNA viroids that incorporate the hammerhead ribozyme self-cleaving motif. We have recently crystallized this larger, wild-type RNA and have obtained native data sets on two crystal forms at SSRL. We hope now to solve the structure.
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