The long-term objectives of the research plan are to elucidate the structure-function relationships which control the elongation cycle during protein biosynthesis and to develop antibiotics which effectively inhibit translation in infectious bacteria and other rapidly growing cells such as tumors. The primary approach is to study the set of four EF-Tu complexes formed during the elongation cycle in E. coli by a variety of techniques, including X-ray diffraction analysis. One short- term objective is to refine the three-dimensional structure of a modified form of EF-Tu-GDP to a resolution of 2.0 angstroms in order to obtain an accurate description of the location and intramolecular contacts of each amino acid. The structure will subsequently be used in a series of difference Fourier analyses to determine the location of inhibitor and antibiotic binding site(s) on EF-Tu. This latter information will provide the appropriate foundation on which to rationally design new antibiotics using EF-Tu as the target protein. Another goal is to complete the crystallographic analysis of the EF-Tu-Ts complex at 5 angstroms and to extend the analysis to a resolution of 3 angstroms. Other short-term goals include the bulk preparation and crystallization of the E. coli EF-Tu-GMP.PNP and EF-Tu-GTP-aminoacyl-tRNA complexes. The major significance of this research is that the set of EF-Tu complexes is the first model system for understanding and controlling three fundamental mechanisms which not only regulate the elongation cycle but which are mimicked by other normal and abnormal intercellular processes. The most important is the GDP/GTP exchange mechanism which mediates signal transduction across membranes from such important cell- surface receptors as those involved in vision, olefaction, oncogenesis and cell regulation by a host of hormones and neurotransmitters. The second fundamental mechanism is that of selective recognition of noninitiator aminoacyl-tRNAs during elongation and its implication for understanding the molecular basis of protein-RNA interactions in general. The third mechanism is that of protein catalysis and the potential for obtaining an accurate three-dimensional description of the conformational changes that accompany the conversion from the reactant form through the transitional state to the product form.
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