The overall goal of this project is to understand the structural basis for specific interaction in two RNA-protein complexes: the HIV Rev-RRE interaction, and the HTLV Rex-RxRE interaction. The main tool to be applied is multidimensional NMR spectroscopy, supported by a variety of biochemical techniques. In addition, we plan to develop peptide bead libraries as a tool for the study of RNA-protein interactions. 1) Determine the structure of the Rev-Response Element (RRE) RNA in complex with Rev-peptides. Using structural constraints obtained through NMR spectroscopy, we will determine the structure of the RRE while in complex with Rev peptides. 2) NMR analysis of variant RRE RNAs, both free and bound to Rev peptides. Presently, the free form of the RRE RNA does not appear to adopt a unique structure. In order to better understand the structure of the free RNA, conformational changes that occur upon peptide binding, and the structure of the RNA-peptide complex, we will study these variant RNAs using NMR. 3) Determine the optimal peptide sequence for characterizing the peptide- RNA contacts by NMR. The peptides currently under study in the Rev-RRE system are not optimal for NMR characterization of the RNA-peptide complex. We will survey variant peptide sequences, in which non- essential arginine residues have been removed or in which alternative helix-stabilizing methods have been used, in order to identify a peptide better suited to NMR analysis. 4) Determine the peptide structure and peptide-RNA contacts using NMR to fully define the structure of the complex. Once a well-defined peptide RNA complex is found, we will be able to completely determine the structure of the complex. Of particular interest are the hydrogen bonding contacts to the RNA from the side chains, and the conformation of the bound peptide. 5) Biochemically characterize the complex between the HTLV Rex-Responsive element, and Rex protein or peptides. We will begin biochemical characterization of the Rex-RxRE system. This regulatory system is functionally equivalent to the HIV Rev system, but is structurally quite different. The ultimate goal will be to prepare a RNA-peptide model system suitable for NMR studies. 6) Develop peptide bead libraries as a biochemical tool to study RNA- protein interactions. We will implement peptide bead libraries to reduce the burden of peptide synthesis, using the Tat-TAR and Rev-RRE systems as a paradigm where the specificity is understood. If successful, the bead library strategy will be applied to the Rex system (Aim 5).
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