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.
Highlights
From the Department of Microbiology and Molecular Genetics, California College of Medicine, University of California, Irvine, Iruine, California 92717
The level the synthesis of all of the aminoacyl-tRNA synthetases has been shown to be controlled by metabolic regulation, several types of amino acid specific regulation have been documented [5,6,7].Amino acid-specific regulation of an aminoacyl-tRNA synthetase is effected when growing cells encounter a growth rate limiting intracellular
The valS transcriptional unit encodes only the valyl-tRNA synthetase gene since the 3‘ terminus of the amino acid-coding regionthiosfgene is followed phenylalanyl-tRNA synthetase gene is regulated by translational control of transcription termination at an attenuator site preceding the pheS gene which responds to intracellular aminoacylated tRNAPhelevels [7]; and, it has been proposed closely (26 base pairs) by an efficient p-independent that theglnS gene, encoding glutaminyl-tRNA synthetase, is transcription terminationsite
Summary
The specific activity of valyl-tRNA synthetasein the cellular ladder [14]prepared with the same end-labeled HindIII/Sau3AI DNA extracts was determined by measuring the esterification of L-['~C] fragment, were heated for 2 min at 90 "C prior to analysis bygel valine to E. coli tRNA at 37 "C. I n Vitro Transcriptions-The uniformly labeled [a-32P]GTPin EtOH precipitation, washing with 70% EtOH and desiccation, the uitro transcription reactions were carried out as previously described primer extension products were resuspended in 100gl of 0.1 N NaOH [22] utilizing closed circular supercoiledplasmid DNA templates. The [y-3ZP]NTPend-labeled in uitro transcription reactions were xylene cyanol, and 0.1% (w/v) bromphenol blue and analyzed by performed as follows.All four [y-32P]NTPsutilized were prepared electrophoresis in a 6% polyacrylamide, 8 M urea denaturing gel.
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