STRUCTURAL INVESTIGATIONS OF RIBOZYMES

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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 been investigating structural aspects of catalysis by RNA enzymes. Our current work focuses on the group I intron and the metabolite responsive ribozyme, GlmS. The GlmS ribozyme is activated by the small sugar, glucosamine-6-phosphate (GlcN6P). This ribozyme is classified as a riboswitch, as the cleavage event results in the down regulation of GlcN6P production in many Gram positive bacteria. The precise chemical mechanism by which the RNA structure of the GlmS ribozyme promotes self cleavage is unknown. Does this nucleolytic ribozyme utilize catalytic metal ions, such as with the group I intron, or does it employ one or more of the heterocyclic bases for general acid or general base catalysis, such as the HDV ribozyme? Does the GlcN6P induce a conformational change to promote an activated RNA fold, or does one of its functional groups, such as the amino group, participate directly in the chemical reaction? If the latter is the case, it would be the first example of an RNA that utilizes a small molecule cofactor to promote chemistry. Interestingly, the amino group of GlcN6P is absolutely required for activity and the pKa of this amine varies with the pKa of the ribozyme. The crystal structure of this ribozyme would provide significant insight into these biochemical questions. It would also provide a structural framework for designing and interpreting all subsequent biochemical studies of this RNA.