RNA functions as an informational, structural and catalytic molecule in cells. However, in most if not all cases where RNA serves as an essential component of a cellular machine, the RNAs collaborate with protein cofactors. Cellular ribonucleoprotein machines for which there is strong evidence that essential active site structures are provided by an RNA include group I and group II introns, RNase P, the spliceosome, the ribosome and telomerase. This non-exhaustive list emphasizes that, although cellular RNA-protein catalysts are relatively rare, they carry out vital processes including RNA biogenesis, polypeptide synthesis and chromosome maintenance. The roles for the protein cofactors have just begun to be explored but include stabilizing and modulating RNA architecture, promoting domain assembly and disassembly, increasing specificity, and allowing regulation. The ultimate goal of this project is to provide insight into the functioning of cellular machines composed of RNA and protein subunits. A one protein-one RNA ribonucleoprotein catalyst composed of the fifth intron of cytochrome b pre-mRNA (the bI5 group I intron) and CBP2 protein (cytochrome b pre-mRNA processing protein 2) from Saccharomyces cerevisiae will serve as a model system. In this simple system, the RNA component contains the active site for the splicing reaction while the protein component enhances the rate of splicing by holding the RNA in its active conformation. The goal of this project, a collaboration with Dr. Kevin Weeks? laboratory at UNC Chapel Hill, is to determine the crystal structure of this simple ribonucleoprotein catalyst. A deeper understanding of this simpler system is likely to yield important generalizations in the broad areas of RNA-protein interactions and protein-facilitated RNA catalysis. Additional information: CBP2 protein can be produced in E. coli and approximately 20 mg of pure protein can be obtained per liter of culture. Conditions for in vitro transcription of the bI5 RNA have been optimized and approximately 0.6 mg of RNA can be obtained per ml of in vitro transcription reaction. Stoichiometric protein:RNA complexes can be formed and analyzed by gel filtration chromatography. Crystallization trials of protein:RNA complex are ongoing.
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