Formation of ornithine transcarbamylase in cells and protoplasts of Escherichia coli

Abstract

Requirements for the synthesis of ornithine-transcarbamylase activity in Escherichia coli W cells and protoplasts upon release from arginine suppression were studied. Results show that the appearance of OTC activity corresponds to the formation of new protein. A complete inhibition of OTC formation resulting from treatment with chloramphenicol lends additional support to the contention that OTC activity appears concurrently with new protein biosynthesis. Protoplasts prepared with lysozyme seem to magnify requirements for OTC synthesis, as evidenced by the necessity for a complete complement of amino acids and magnesium for full capacity synthesis of OTC by protoplasts. A curious and as yet unexplained by-product of protoplasts formation was the partial loss of arginine's ability to supress OTC synthesis. The following observations suggest a role of RNA in OTC synthesis: RNase inhibition of protoplasts, stimulation by uracil of the uracil-requiring mutant, and stimulation by purines and pyrimidines of both cells and protoplasts. Investigations with a uracil-requiring mutant of E. coli revealed that the burst of OTC formation after release from arginine suppression could be obtained in the absence of uracil. In direct contrast, the presence of uracil was essential for the induction of β-galactosidase by this uracilless mutant. Experiments in which inorganic 32PO 4 was incorporated into RNA nucleotides of the uracilles mutant during OTC synthesis indicated that new RNA synthesis was negligible in the absence of uracil. The experimental evidence suggests that performed enzyme-forming sites, held under arginine suppression, commence manufacture of OTC molecules immediately upon release from arginine suppression. It seems likely that, under these conditions, synthesis of this enzyme occurs without new RNA formation.