The PI plans to use the TAT-TAR system for development of techniques to probe the 3-dimensional structure of protein-RNA complexes using a variety of novel physical and chemical approaches. He has developed techniques to link chelators to unique nucleotides in chemically synthesized RNA that permit determination of nearby residues by indentification of the sites of cleavage mediated by the metal-chelator conjugate. This technology will also be used to monitor conformational changes that occur upon binding of protein factors. Specific residue contacts in bound proteins iwll be determined by ribonucleotide-linked celators that serve as proteases, through photo-crosslinking and through the use of chelating amino acid analogs inserted into specific sites of TAR binding TAT peptides. In this latter approach contacts will be determined by clelate-mediated cleavage of the bound RNA. The PI will also convert chelators bound to specific RNA sites to fluorescence energy donors for transfer to acceptors attached to Tat peptides. The distances determined by these methods will be used to set constraints on possible RNA-protein structures.
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