HIV gene expression is regulated by the viral protein tat which acts through an RNA regulatory element (TAR) to markedly enhance transcription. This RNA is thought forms a stem-loop structure which mediates the trans- activation event. There is currently widespread interest in understanding the details of this process. While various aspects of the trans-activation event have been characterized in cells and tat has been shown to bind TAR RNA in vitro, our understanding of the structure and dynamics of these molecules and the chemical basis for recognition remains limited. The goal of this proposal is to develop new spectroscopic and chemical approaches which allow the precise atomic interactions required for complex formation to be identified, and their energetic contribution to binding assessed.
The specific aims are to: 1) develop new fluorescence-based methods for exploring the energetics and dynamics of tat-TAR complex formation. 2) identify the precise nucleotide components in TAR RNA which make important binding interactions with tat. 3) synthesize a series of TAR analogs containing chemically or photochemically reactive functional groups which can be used as affinity reagents to identify residues in tat which interact with TAR RNA, and 4) critically evaluate the proposed secondary structure of TAR RNA using NMR and to determine how mutations in the stem-loop as well as tat binding alter its conformation and dynamics. Emphasis will be placed on the use of fluorescence polarization and radiationless energy transfer to measure the kinetics of tat-TAR complex formation to in solution. This will allow the specificity of the interaction to be rigorously examine using reagents and approaches developed in Aims 2 and 3. One approach will involve the analysis of atom-scanning mutants of TAR RNA, derivatives in which a single atom or substituent contained on a particular nucleotide ring implicated in tat binding is replaced by hydrogen or otherwise modified. By disrupting a single binding interaction (H-bond) and characterizing the energetic contribution of the deleted atom or substituent to complex formation, it will be possible to map the interaction surface on TAR RNA. Coupled to this approach will be the development of affinity labeling TAR derivatives useful int he identification of residues in tat which interact directly with specific nucleotides in the RNA. NMR experiments (Aim 4, in collaboration with Dr. Garry King, Rice University) will provide new information concerning the conformation and dynamics of TAR RNA and will shed light on atomic changes which result upon tat binding. The successful completion of any of these aims will constitute a significant advance in our understanding of the chemical basis of tat-TAR binding and could facilitate the design of agents which block trans-activation in vivo.
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