The long term objectives of the proposed research is to determine the molecular basis of the transport of amino acids in bacteria. Cloning and DNA-sequencing techniques are being used to isolate and to characterize the five components of the branced chain amino acid transport systems in Escherchia coli. Two of the leucine transport components, livJ and K, are periplasmic binding proteins and three additional components, livH, M, and G, are membrane associated. The current studies will concentrate on three areas. First, M13 cloning and nucleotide sequencing techniques will be applied to determine the DNA sequence of the membrane components. The expression of the membrane components will be amplified by providing high efficiency controllable promoters for the cloned livH, M, and G genes. Gene fusion techniques to produce hybrid proteins are being carried out to facilitate the preparation of antibodies to the membrane components. These antibodies will be used for their purification and characterization. The study of the regulation of leucine transport constitutes a second specific goal of this proposal. The repressor components, livR and 1stR, will be isolated and characterized with the aid of cloning and DNA sequencing techniques. A study of the role of rho factor dependent transcription termination in the regulation of leucine transport will be carried out. A third specific goal is a study of the protein export in bacteria using the periplasmic leucine binding proteins as a model system. The structural and energy requirements for export of proteins into the periplasmic space will be determined using directed mutagenesis techniques. In addition, vectors for obtaining the export of other proteins fused to the leucine specific binding protein will be constructed. Finally, the introduction of oligonucleotide-directed mutagenesis techniques will facilitate structure-function studies with several of the leucine transport components.
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