Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB).
The Bacillus subtilis gene (pyrB), which encodes aspartate transcarbamylase (ATCase), was cloned on a HindIII restriction endonuclease fragment inserted into the pUC13 plasmid vector. B. subtilis pyrB was expressed in Escherichia coli, as judged by complementation of E. coli pyrB mutants and production of enzyme that was specifically inhibited by antibody directed against B. subtilis ATCase. The extent of expression was strongly dependent on the orientation of the inserted DNA in the vector, which suggested that transcription was initiated from vector-borne (rather than B. subtilis) promoters. The entire 1098-base pair HindIII fragment of B. subtilis DNA was sequenced by the Maxam-Gilbert method. The amino acid sequence of B. subtilis ATCase was deduced from a 305-codon open reading frame and agreed very well with analyses of the purified enzyme. Comparison of the sequence of B. subtilis ATCase with that of E. coli ATCase catalytic subunit, for which the three-dimensional structure is known, revealed many homologous residues of probable importance in catalysis and structural folding of ATCases. The significance of homology to E. coli ornithine transcarbamylases was also analyzed. The sequences of the 5' and 3' flanking regions to pyrB encode open reading frames in both cases which overlap with pyrB by eight and six codons, respectively. It is probable that these open reading frames encode other enzymes of a coordinately regulated unit. The sequence 5' to pyrB also encodes an mRNA bearing a pyrimidine-rich sequence followed by a typical sequence for a rho-independent transcription terminator. The presence of these elements and the 5' open reading frame suggest that B. subtilis pyrB, like E. coli pyrBI, is regulated by an attenuation mechanism.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism
D000596:Amino Acids; D004269:DNA, Bacterial; D012333:RNA, Messenger; D001221:Aspartate Carbamoyltransferase; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII