To achieve its diverse range of biological functions, RNA must fold into specific tertiary structures that create active sites for chemical transformations or recognition features for protein binding. Conformational transitions necessary to achieve biological activity involve large movements of helices, loops and other structural elements. It is difficult to study these dynamic conformational transitions using conventional methods of structure analysis. Time-resolved fluorescence resonance energy transfer (tr-FRET) is a powerful technique for the study of RNA conformational transitions because it can provide structural, thermodynamic and kinetic information. In this proposal, tr-FRET and related spectroscopic methods will be used to study biologically-relevant RNA conformational transitions in three different systems.
The specific aims are: 1. Analyze tertiary structure formation in the hairpin ribozyme. Elucidate the structural and thermodynamic basis for efficient docking of the substrate-binding and catalytic domains of the ribozyme-substrate complex and characterize changes in base pairing and stacking that occur during domain docking. 2. Elucidate the energetic basis for ion- and protein-induced folding of RNA three-way junctions. Determine the global conformations of three-way junctions from 16 S and 5 S rRNAs and establish how the binding of metal ions and proteins are linked to conformational changes within the RNA. 3. Determine the global structure of a large RNA fragment from the Rev Response Element. Establish whether binding of HIV-1 Rev and Rev-Rev multimerization on the RNA induce a rearrangement of helices. The results of this research will reveal how the structure of a helical junction can direct the docking of distant RNA domains and stabilize a biologically active tertiary structure. The findings will also contribute to an understanding of how proteins and metal ion cofactors can regulate the biological activity of RNA. In addition, the results will aid in the design of improved therapeutic ribozymes and contribute to the development of drugs directed against RNA targets.
