Project 2 Enzymatic activity and structure probing

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RNA molecules, like proteins, fold into specific three-dimensional structures that are required for their biological function. However, our understanding of how RNA molecules attain their native conformations is in its infancy. Prior studies on the structure, mechanism, and folding of the Tetrahymena group I ribozyme render it an ideal RNA for further dissection of the folding behavior and of the molecular and topological properties responsible for this behavior. This Component Project invokes several approaches, some well-established, others straightforward modifications of well-established techniques, and some novel approaches, to address the nature of the observed rapid electrostatic collapse of this RNA, the structures and properties of folding intermediates on established and new folding pathways, and the molecular features that govern the rates of interconversion between species. Techniques include time-resolved fluorescence intensity measurements with ribozymes containing 2-aminopurine site-specifically incorporated, time-resolved chemical probing of RNA structure, mutagenesis, introduction of topological constraints by helix extensions, surface tethering, and site-specific cross-linking, and the use of binding and activity measurements to assess the properties of intermediates, kinetic partitioning events during the folding process, and the folding outcome. These and the data from the other Component Projects, when integrated by the Computational Core, will greatly further the Tetrahymena ribozyme as a paradigm for understanding the folding and dynamic behavior of RNA. This understanding will provide a basis for comparative studies with other RNAs and protein.RNA complexes, as well as an opportunity to learn more about the differences and similarities in the basic behavior of RNA and protein macromolecules. An understanding of the fundamental behavior of RNA provides a starting point for determining how its behavior may be altered, controlled, or augmented by cellular interactions and in therapeutic intervention. Knowledge of these fundamental properties will provide a foundation for studies focusing on the cellular behavior of RNA, on the role of RNA in disease, and on the potential use of RNA as a drug target or therapeutic.