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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM011024-25
Application #
3268220
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1976-06-01
Project End
1990-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
25
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Haney, S A; Platko, J V; Oxender, D L et al. (1992) Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli. J Bacteriol 174:108-15
Haney, S A; Oxender, D L (1992) Amino acid transport in bacteria. Int Rev Cytol 137:37-95
Williamson, R M; Oxender, D L (1992) Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli. J Bacteriol 174:1777-82
Su, T Z; Schweizer, H P; Oxender, D L (1991) Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli. Mol Gen Genet 230:28-32
Adams, M D; Maguire, D J; Oxender, D L (1991) Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli. J Biol Chem 266:6209-14
Adams, M D; Oxender, D L (1991) Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli. J Cell Biochem 46:321-30
Williamson, R M; Oxender, D L (1990) Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli. Proc Natl Acad Sci U S A 87:4561-5
Adams, M D; Wagner, L M; Graddis, T J et al. (1990) Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli. J Biol Chem 265:11436-43
Nazos, P M; Antonucci, T K; Landick, R et al. (1986) Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli. J Bacteriol 166:565-73
Antonucci, T K; Wagner, L M; Oxender, D L (1986) Cloning, expression, and nucleotide sequence of livR, the repressor for high-affinity branched-chain amino acid transport in Escherichia coli. Proteins 1:125-33

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