The broad, long-term objective of this proposal is to gain structural and dynamic insights into how proteins modulate RNA structure and function in a catalytic ribonucleoprotein (RNP) complex. Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs. The eubacterial enzyme is an RNP complex made up of one RNA and one protein subunit. The RNA subunit is catalytic on its own, but the protein is required in vivo. In contrast, several protein subunits with unknown function constitute the human enzyme, and its RNA component alone appears to be inactive. The investigators propose that a principal role of the protein subunits is to stabilize the active conformation of the RNA, and that holoenzyme assembly and substrate recognition proceed via hierarchical mutual binding-induced folding of the protein and RNA subunits. Using the RNase P enzyme from a thermophilic archaebacterium as a model, the investigators will use biochemical and biophysical tools to gain structural insights into the interaction between individual protein components, free and in complex, with the catalytic RNA subunit.
The aims are to: 1. Determine the solution structures of one or more protein subunits (or their fragments) by NMR. 2. Map the secondary structure and protein binding sites of the RNA subunit using enzymatic and chemical probes. 3. Characterize protein-protein and protein-RNA interactions in the native and in vitro reconstituted enzyme, and examine whether conformational changes (induced fit) accompany RNP assembly. Successful completion of these aims will enable integration of the data into a three-dimensional model of the RNase P holoenzyme and provide valuable insights into how proteins modulate the enzyme's fUnction. With recent efforts to make use of the substrate selectivity of RNase P to engineer customized ribozymes for use in gene therapy, insights into the molecular basis for the enzyme's function is clearly essential.
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