Time Resolved Hydroxyl Radical Footprinting of RNA

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DESCRIPTION (provided by applicant): RNA molecules and RNA-protein complexes are dynamic structures, and this motion is intrinsic to their function. The study of RNA folding mechanisms will provide physical insight into the assembly of RNAprotein complexes during gene regulation, and conformational changes that take place during catalysis. Although RNA secondary and tertiary interactions form quickly, some RNAs are easily trapped in misfolded states. Competition among alternative conformations can lead to disease if RNAs malfunction, or can be exploited for regulation of gene activity and replication of RNA viruses. Thus, the ability to understand and predict RNA function from its primary sequence is important to the analysis and treatment of genetic disease, and the development of therapeutic agents that target RNA molecules. The folding mechanism of a small group I ribozyme from Azoarcus will be compared with folding of the larger Tetrahymena ribozyme. This is an ideal system to study early steps in the assembly of RNA structure, because more than half the RNA folds within 100 ms, and the tertiary structure is stablilized by many types of metal ions. Hydroxyl radical footprinting using a synchrotron X-ray beam will be used to detect changes in RNA tertiary structure at 20 ms intervals. This method is unique, in that it resolves conformational changes at specific sites in the RNA. Stopped-flow fluorescence spectroscopy will be used to monitor global folding of RNAs containing 2-aminopurine and fluorescein, with 1 ms resolution. The proposed studies address how sequence and metal ion interactions direct the assembly of specific tertiary structures native conformations. Time resolved hydroxyl radical footprinting will also be used to study the assembly of RNA-protein complexes, using the bacterial RNase P holoenzyme as a test system. RNase P is an essential enzyme present in all cells. The folding pathway of the RNA subunit shares many properties with those of group I ribozymes, but it is not known how the protein subunit alters the formation of catalytically active RNA. As Xrays easily penetrate cells and whole tissues, this method will be adapted to study the three-dimensional structure of group I riboyzmes and RNase P in vivo.