The biological function of RNA is intimately related to its three-dimensional structure. The objectives of this proposal are to understand how the sequence and structure of an RNA determines its folding pathway and thermodynamic stability, and to elucidate how ribozymes fold during transcription, a mimic of in vivo folding behavior. The catalytic RNA component from bacterial ribonuclease P will be used throughout this proposal. The catalytic, C-domain of this ribozyme folds without kinetic traps, providing an ideal system for the analysis of tertiary RNA folding. Aim I focuses on how the C-domain builds its structure over time, and identifies critical features of the limiting step. To explore the strategies employed by natural RNAs to modulate their stability, Aim 2 will identify the minimal number of sequence changes necessary to create a thermophilic ribozyme from its mesophilic homologue. Aim 3 extends our investigation of the principles of ribozyme stability to another class of RNase P RNAs that have the same catalytic core, but different peripheral regions. Aim 4 is to test whether species-specific pausing during transcriptional elongation is a mechanism for enhancing folding during transcription. This broad research program will deepen our understanding in how RNA folds into its tertiary structure and establish the fundamental principles of RNA folding.