Research Plan: The long-term goal of the applicant is to understand the mechanism of gene regulation in HIV. The application presents a plan to investigate the interactions of the regulatory factor TAT with the Trans-activation response region of RNA (TAR) in HIV-1. The procedures are based on new chemical methods for the site-specific modification and cleavage of peptides developed by Dr. Rana that allow for detailed studies on protein-protein and protein-nucleic acid interactions. The investigator plans to study the structure of the Tat/TAR RNA-protein complex of HIV using a series of novel chemical and physical approaches. He proposes to attach derivatives of the metal chelator EDTA to various positions in the TAR RNA molecule, induce RNA cleavage by addition of iron or copper, and then map the sites of cleavage by gel electrophoresis. He will compare the auto-cleavage patterns of TAR and the Tat-TAR complex, to detect structural changes induced in TAR by Tat binding. Chelate-derivatized TAR RNA also will be used to cleave the Tat protein via the metal-catalyzed proteolysis reaction discovered by Dr. Rana as a graduate student, in order to map the amino acids of Tat that are near the site of chelate attachment in TAR. In a reciprocal experiment, investigator plans to site-specifically derivatize the Tat protein with a metal chelator, and then map the sites of TAR cleavage induced by modified Tat. Fluorescence energy transfer experiments will be used to measure the distance between a lanthanide donor bound to a chelate-derivatized TAR RNA molecule, and a fluorescent dye acceptor attached to various sites in Tat.
