The role of ribosomes in the cell-free formation of tryptophan synthetase in Escherichia coli

Abstract

An increase in activity of B protein of tryptophan synthetase ( l-serine hydro-lyase (adding indole), EC 4.2.1.20) was observed when either of two ribosomal fractions from Escherichia coli was incubated with a supernatant fraction under appropriate conditions. In the “P 30 system” involving larger ribosomes, the activity increase was sensitive to inhibition by DNAase (EC 3.1.4.5), RNAase (EC 2.7.7.16), chloramphenicol, streptomycin and by l-tryptophan. In contrast, the “P 60–180 system”, containing smaller ribosomes, was insensitive to DNAase, chloramphenicol and streptomycin and partially sensitive to RNAase and l-tryptophan. Experiments in which a ribosomal or supernatant fraction from a deletion mutant was substituted for the corresponding wild-type fraction clearly demonstrated that the wild-type supernatant is essential for an activity increase in the P 30 system, whereas the wild-type ribosomes are required in the P 60–180 system. These results indicate the fundamental difference between the two systems with respect to the underlying mechanisms. The evidence suggests that the P 30 system involves a DNA-dependent formation of the enzyme, while the P 60–180 system exhibits an “activation-and-release” of nascent enzyme molecules from ribosomes. Furthermore, the latter reaction may represent a part of the mechanism leading to the increase in enzyme activity in the P 30 system. Implication of these results and the role of ribosomes in enzyme formation will be discussed.