EXCEED THE SPACE PROVIDED. Group II introns are ribozymes with a diversity of biological functions: They are self-splicing RNA molecules that also behave as transposable elements. Their mobility among genomes is one plausible explanation for the dispersal of introns throughout eukaryotic organisms and they are widely believed to represent an ancestral form of the eukaryotic spliceosome. Research on group II introns therefore provides important insights into general splicing mechanisms and is leading to the development of targeted introns for potential application in biotechnology and gene therapy. At a more basic level, group II introns have an unusual architecture that is providing a wealth of l information on RNA folding and tertiary structure. In order to apply group II introns in biomedical land basic research, and to better understand how they function in splicing and mobility, we must be lable to visualize their three-dimensional architecture. It is particularly important to understand the Istructure of the intron active-site, branch-site and surrounding catalytic core. To this end, we propose a series of experiments designed to elucidate the spatial organization and function of critical group II intron domains 1,2, 5 and 6, by emphasizing a combination of chemogenetic methodologies (nucleotide analog interference suppression), site-directed crosslinking, molecular modeling and crystallography. A diverse family of group II introns will be investigated in order to exploit the unique thermostability, ionic requirements and mechanistic features that are provided by individual examples. By combining biochemical and biophysical methods in this manner, we hope to obtain structural information that reflects active and biologically informative conformations of the ]roup II intron. PERFORMANCE SITE ========================================Section End===========================================
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