The long term objectives of this research program are to contribute to the understanding of structure and function in RNA molecules by determining principles of metal-RNA interactions. The importance of RNA in controlling genetic information, and potential for us in Gene therapy applications, drive considerable current interest in the physical and chemical foundations of RNA structure and chemical reactivity. Metal-RNA interactions are critical in stabilizing specific RNA tertiary structures, and in participating in RNA-catalyzed chemical reactions. The properties of metal sites in RNA are fundamental to the relationship between structure and reactivity, and increased knowledge of metal-RNA interactions ma aid in the design of more efficient RNA catalysts, for example to be used as therapeutic agents in vivo. A novel emphasis of this program concerns using spectroscopic techniques to obtain specific information about metal coordination sites in catalytic RNA molecules, or ribozymes. The study of metals in proteins has long made intensive use of metal-based spectroscopic methods, which provide information about numbers and types of binding sites as well as identification of specific metal ligands. For RNA molecules, recent advances in large-scale sample preparation, an ever-increasing body of knowledge from biochemical experiments and intriguing predictions from X-ray crystallography studies make such spectroscopic studies both feasible and timely.
Specific aims of this work involve measuring metal-RNA interactions in large Group I intron form Tetrahymena thermophilia. This ribozyme catalyzes a metal-dependent self-splicing reaction, can be separated into two stable subdomains, and provides a relatively well-studied model system that is expected to have a variety of different metal ion sites. The number and affinity of divalent cation binding sites in the group I intron and its subdomains, metal ion specificity's, and ligand environments will be pursued using spectroscopic techniques including EPR, NMR, electron-nuclear double resonance spectroscopy, and others as needed. EPR-active spin labels will be used to probe the dynamics of motions in this large RNA system, and to map out metal sites and tertiary interactions. In addition to gaining specific information about the group I intron, these studies will in general elucidate previously unknown properties of large RNA molecules.
Showing the most recent 10 out of 15 publications
