Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II

• Published in: The Journal of biological chemistry 1988-07, Vol.263 (19), p.9526-9532
• Main Authors: Chen, N Y, Paulus, H
• Format: Article
• Language: English
• Subjects: Aspartate Kinase - genetics; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genes, Bacterial; Genes. Genome; Genetic engineering; Genetic technics; Genotype; Macromolecular Substances; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Phosphotransferases - genetics; Plasmids; Synthetic digonucleotides and genes. Sequencing; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)76574-0

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.