The proposed research will develop a general, biochemical method for the determination of tertiary contacts within complex, biologically relevant RNA structures. The long term goal of this project is to use this new method for the elucidation of tertiary structures of multi-subunit RNA complexes, such as ribosomes and spliceosomes, whose size precludes study in the near future by x-ray or NMR techniques. The methodology will be developed using the hammerhead ribozyme as a model system and subsequently applied to the hairpin and the Neurospora versus ribozymes.
The specific aims are: (1) To incorporate chemical nucleases and cross-linkers at; -positions of specific nucleotides in helices I and II of the hammerhead ribozyme. As these helices are known to be proximal in the active structure, this will allow the evaluation of the reactive probes for determining RNA tertiary interactions. (2) To determine the usefulness of this method for studying RNA folding pathway by applying it to the hammerhead ribozyme which has been shown to undergo a drastic re- orientation of its helices upon titration of magnesium ions into the solution. (3-4) Having established tools for probing interactions between elements of secondary structure, to study the structures of the hairpin ribozyme, in collaboration with Prof. E. Westhof, and the Neurospora versus ribozyme, in collaboration with Prof. R. Collins. These structural studies will increase of mechanistic understanding of ribozymes and may lead to the design of improved RNA-catalysts. This has therapeutic implications as ribozymes hold promise for antisense treatment of oncogenic and viral diseases. The proposed method should also be applicable for the determination of tertiary contacts between regions of non-catalytic RNAs and could be extended to the investigation of DNA and RNA protein interactions. Thus, the results of the proposed experiments will be of interest to those working in the areas of gene therapy, Rna structure, biomolecular catalysis and molecular recognition.
