VALYL-TRANSFER RNA-SYNTHETASE GENE OF ESCHERICHIA-COLI-K12 - MOLECULAR GENETIC-CHARACTERIZATION

Abstract

We report the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene which encodes the enzyme valyl-tRNA synthetase (EC 6.1.1.9). The valS gene was subcloned from the Clarke-Carbon plasmid pLC26-22 by genetic complementation of the valS temperature-sensitive mutant strain, AB4141. The protein-coding region of the valS structural gene was determined by in vitro DNA directed coupled transcription-translation assays. Assays of cellular extracts of cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase-specific activity 12-fold. The DNA sequences of the 5''- and 3''-terminal regions of the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both .alpha.-32P labeled and .gamma.-32P-end-labeled in vitro transcription assays. In vivo, valS transcription initiates from tandem overlapping promoters separated by seven nucleotides. In vitro, only the upstream promoter is active. The presence of several regions of hyphenated dyad symmetry overlapping the tandem promoter region are noted. The valS translational start codon (AUG) is located 93 base pairs downstream from the major transcription initiation site. The valS transcriptional unit encodes only the valyl-tRNA synthetase gene since the 3'' terminus of the amino acid-coding region of this gene is followed closely (26 base pairs) by an efficient .rho.-independent transcription termination site.