The goal of the proposed research is to exploit the hairpin ribozyme and the power of mechanistic enzymology to develop a detailed, quantitative view of the kinetics and equilibria of RNA-mediated reactions in living cells. RNAs are critical components of the biological machinery responsible for maintenance and expression of genetic information, providing potential targets and reagents for therapeutic intervention. The dynamics and equilibria of RNA conformational transitions play key roles in transcription, translation and RNA processing reactions. Determining the functional significance of specific structures and conformational transitions in RNA-mediated biological processes is complicated by the participation of many components in complex pathways. On the other hand, mechanistic studies of RNA enzymes in vitro have produced detailed, quantitative descriptions of the kinetics and equilibria of individual steps of RNA-mediated reactions and the influence of small molecules, RNA binding proteins, and RNA transcription on RNA assembly pathways. The hairpin ribozyme provides an especially valuable model system for RNA structure-function studies in vivo because ribozyme variants can be designed so that intracellular cleavage activity monitors specific RNA conformational transitions along the reaction pathway. Mechanistic insights gleaned using this simple system to report on RNA structure and dynamics in specific biological contexts will help illuminate mechanisms of more complex RNA-mediated reactions and provide a rational framework for the development of RNA-based therapeutics. Specific goals include: establishing a kinetic and thermodynamic framework for mechanistic studies of RNA conformational transitions in vivo, determining whether RNA structures display different conformational equilibria and dynamics in nuclear and cytoplasmic compartments, determining how transcription and translation influence RNA assembly pathways in vivo, characterizing allosteric regulation of RNA structure and function by small molecule and protein effectors in vivo, and developing a system for ribozyme-mediated mRNA repair through sequential cleavage and ligation reactions. Mechanistic insights gleaned using the hairpin ribozyme to report on RNA structure and dynamics in specific biological contexts will help illuminate mechanisms of more complex RNA-mediated reactions and provide a rational framework for the development of RNA-based therapeutics.