The main focus of the proposed research is to understand the nature of interactions between the regulatory factor, Tat, and trans-acting responsive region of RNA (TAR) in human immunodeficiency virus-1 (HIV-1), and determine the structure of the Tat-TAR complex under physiological conditions. Knowledge of the structure of the protein-RNA complex, Tat-TAR, would greatly improve our understanding of the function of this complicated regulatory system. Structural information about the Tat-TAR complex will be obtained by using metal chelates site-specifically attached to RNA and peptides. Metal-catalyzed oxidative reactions will be initiated by the addition of redox-active metal salts and reducing agents in the presence of oxygen or hydrogen peroxide. These reactions cause the oxidative cleavage of RNA in the vicinity of the metal site. Chemical and enzymatic analyses will be carried out to locate the cleavage sites in RNA, and this information will reveal which bases in the folded RNA were close to the metal site. RNA labeled with metal chelates will be used to understand the three-dimensional folding of RNA in the presence and absence of the RNA-binding protein, since the protein binding causes a change in the tertiary structure of RNA and cleavage sites in the RNA sequence may differ in the presence or absence of RNA binding proteins. Interaction of protein side chains with RNA will be investigated by two methods: (a) RNA-binding peptides will be labeled site-specifically with metal chelates, and these peptides will be used to cleave RNA by metal-catalyzed oxidative reactions. Results from the analysis of the cleavage products will provide information about the protein contact sites on RNA. (b) Site-specifically labeled metal chelates can also serve as donors for fluorescence energy transfer experiments when lanthanide metal ions are used instead of redox active metals. Metal chelates which are incorporated into RNA site-specifically will be used as donors for energy transfer experiments. RNA-binding peptides will be labeled with fluorescein, acceptor molecules for energy transfer. The fraction of energy transfer will be used to determine the distance between peptide side chains and the metal site in RNA. The long term objectives of this project are to develop powerful and generally applicable methods to design RNA-binding peptides and metal complexes for RNA targeting. Computer modeling will be used to create the structure of TAR RNA compatible with the results of metal-catalyzed cleavage and fluorescence energy transfer experiments. On the basis of obtained structural data, small RNA-binding peptides will be synthesized, and these peptides will be used to deliver catalytic and efficient RNA-cleaving macrocyclic complexes to the target sites.
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