Escherichia coli DNA-directed beta-galactosidase synthesis in presence and absence of Ca2+

Abstract

DNA-dependent synthesis of beta-galactosidase was optimized in extracts made from cells lysed by a standard French pressure cell. Extracts made at 3200 psi synthesized up to 25-fold more beta-galactosidase than extracts made at 7500 psi. beta-Galactosidase synthesis was cyclic 3', 5' AMP dependent, as expected, and in optimal conditions transcription and translation proceeded at 8.6 nucleotides and 2.7 amino acids per s, respectively. The high pressure extracts were stimulated 3- to 5-fold by Ca2+, especially at low Mg2+ concentrations. In contrast, extracts prepared at low pressure were inhibited as much as 50-fold by Ca2+ ions. The inhibition by Ca2+ was analyzed further. Addition of kasugamycin, an antibiotic that acts on ribosomes, to reactions containing Ca2+ stimulated beta-galactosidase synthesis to nearly control levels. Extracts from a kasugamycin resistant mutant were neither inhibited by Ca2+ nor stimulated by the addition of kasugamycin to in vitro reactions containing Ca2+. The change in the mutant was ascribed to the ribosomes by testing combinations of soluble proteins, ribosome wash, and ribosomes from parental and mutant strains. These results suggest that Ca2+ ions inhibit translation by ribosomes, very likely at an initiation step; and that they enhance enzyme synthesis only in conditions where translation is inefficient (high-pressure extracts at low concentrations of Mg2+, for example). This latter effect is probably a consequence of increased RNA stability in the presence of Ca2+ (Cremer, K., and Schlessinger, D. (1974), J. Biol. Chem. 249,4730).