Molecular cloning and characterization of the gene for Escherichia coli valyl-tRNA synthetase.

A ColE1 hybrid, pB2, carrying the structural gene for the valyl-tRNA synthetase, valS, and the pyrBI operon was isolated from the Clarke-Carbon collection. Strain GRB238 with a temperature-sensitive mutation in the valS gene showed a three-fold increase in valyl-tRNA synthetase activity after transformation with pB2. The valS gene was subcloned into pBR322 after partial Sau3A digestion of pB2. The new plasmid, pHOV1 had a 9.4-kb insert in pBR322 and cells carrying pHOV1 showed an eight-fold increase in specific valyl-tRNA synthetase activity. The localization and direction of transcription of valS in pHOV1 was determined by transposon-directed mutagenesis and analysis of protein synthesis in minicells. The valS gene was shown to be transcribed opposite to the direction of DNA replication, i.e., counterclockwise.