The """"""""RNA folding problem"""""""" is a fundamental and challenging question in contemporary biophysics. In addition, studies of folding of macromolecules are presently of great general interest. Understanding the mechanism(s) by which RNA molecules fold into compact structures capable of biological activity is important because RNA folding is closely tied to cellular regulation and metabolism and catalytic RNAs are potential reagents for gene therapy. Unlike the """"""""protein folding problem"""""""" which has been under study for many decades, the study of RNA tertiary structure stability and folding is a relatively new field of endeavor. Thus, an understanding of both the thermodynamics and kinetics of RNA folding are only now beginning to emerge. The proposed equilibrium and time-resolved folding studies of the 385 nucleotide L-21 Sca I Tetrahymena thermophila ribozyme will yield a thermodynamic and kinetic picture of the formation of the individual tertiary contacts that stabilize this large catalytic RNA. Many of these tertiary contacts represent motifs that are present in a wide variety of RNA structures and have high-resolution crystallographic data available for interpreting the relationships of structure and stability. Using time-resolved synchrotron x-ray footprinting we can follow the time-resolved folding kinetics of this large RNA with millisecond time resolution and very high structural resolution. This provides a powerful structural probe of the dynamics of folding for this large RNA. The examination of the ion dependence of folding and the use of chemical denaturants to examine the folding allow a detailed portrait of the stability of individual tertiary contacts to be painted. In addition, the separate examination of independently folding domains and selected mutations will pinpoint the intra-domain and inter-domain tertiary contacts crucial to stability.
