In this applications, studies are proposed to continue a productive investigation of the interactions of HIV RT with the priming tRNA3lys. In the last funding period, Dr. LeGrice's lab identified p51 mutants that, when reconstituted with wild type p66 displayed severely reduced affinity for tRNA3lys and failed to extend tRNA3lys hybridized to the viral genome. (1) In the current application, the hypothesis that these mutants cannot destabilize tRNA-template interactions outside of the PBS, a putative necessary step for elongation, will be pursued. This hypothesis will be examined with deleted viral RNA and tRNA variants to determine whether synthetic capacity can be restored to the mutant through artificial destabilization of specific tRNA-template interactions. (2) Mutagenesis of p51 will be extended to further refine residues required for binding and utilization of the primer. Protein footprinting studies will be conducted on p66 to test the hypothesis that the p51 deletion defects alter the 'connection' subdomain of p51, subtly altering the subunit contact with p66. (3) Chemical and enzymatic footprinting methods to investigate how truncating the p51 connection subdomain influences interaction with the tRNA primer will be continued. Binary RT-tRNA and ternary RT-tRNA-viral RNA complexes will be examined. (4) Synthetic nucleocapsid protein will be examined to determine whether it can complement RT mutants unable to support (-) strand DNA synthesis from tRNA primers. Experiments will attempt to distinguish contributions from protein-protein and protein-nucleic acid interactions. (5) Experiments will be conducted to see if priming tRNA has an allosteric effect in the elongation reaction during the first phase of (-) strand synthesis. (6) Structural studies will be continued including neutron scattering, electron microscopy of 2D crystals (with image reconstruction) and attempted crystallization for X-ray crystallography of RT-tRNA complexes.
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