NMR STRUCTURE OF ZINC FINGERS SPECIFIC FOR HIV RRE RNA

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DESCRIPTION: RNA-protein interactions are of paramount importance for gene regulation of viruses that cause human disease. The long term goal of this project is to understand the structural basis for the interaction of novel zinc finger proteins with the HIV-1 Rev responsive element (RRE) RNA, the binding site for the critical regulatory protein Rev. This knowledge will be exploited for developing small RRE binding proteins that possess both affinity and specificity in living cells. Using a powerful phage display approach that permits the sampling and scoring of a large number of proteins simultaneously, zinc finger proteins have been isolated which bind specifically to HIV-1 RRE and stem loop IIB. NMR structure determination is used to elucidate the zinc finger structure and specifics of RNA interaction. To enhance and further understand the RNA binding properties of zinc fingers, a design process will use structural information to guide in vitro selection as well as traditional site directed mutagenesis experiments. The molecular details of the interaction between zinc finger proteins and RRE stem loop IIB will be determined by NMR. The immediate goals of the proposal are three fold. First, to understand how these engineered proteins bind their RNA substrates by identifying binding sites and the amino acids responsible for RNA contact. Second, to improve their substrate affinity, Dr. Germann uses NMR structural data for site directed mutagenesis experiments and structure guided phage display selection. Finally, the limits and applicability of zinc fingers as inhibitors of biological processes will be determined by selection of zinc finger proteins against new RNA targets. The inhibitory activity of designed zinc fingers will be determined through in vitro and in vivo assays.