Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli.

Abstract

The 4 genes encoding the components of the high-affinity branched-chain amino acid transport systems in E. coli (livH, livG, livJ and livK) were cloned into .lambda. phage and subsequently into the plasmid vector pACYC184. The presence of the 4 structural genes and their accompanying regulatory regions on the resultant plasmid, pOX1, was confirmed by genetic complementation analysis and by transport studies carried out on the appropriate transformed mutant strains. When pOX1 DNA was used to direct an in vitro transcription/translation system, 4 major polypeptide products were produced. Immunoprecipitation with antibody directed against the LIV-binding protein identified the 2 leucine-binding proteins as products of in vitro synthesis. The binding proteins were produced in precursor forms and had MW approximately 2500 higher than the processed, mature forms. A minicell-producing strain transformed with plasmid pOX1 produced the binding proteins in the processed form.