The regulatory process in the de-repression of enzyme synthesis. Alkaline phosphatase of Bacillus subtilis

Abstract

The kinetics of de-repression of alkaline phosphatase in B. subtilis were studied after the removal of Pi]. Enzyme activity appeared about 10 min. after removal of Pi, whereas ''enzyme-forming potential'' appeared after 6 min. Protein synthesis is not impaired for at least 20 min. or removal of Pi, but RNA synthesis is considerably diminished. Adding chloramphenicol to cells without Pi, just at the time they start to make enzyme-forming potential, does not affect the differential rate of enzyme synthesis compared with total protein. Enzyme-forming potential accumulates to about normal levels in the presence of chloramphenicol, even though peptide-bond formation is inhibited by more than 95 percent. Similar experiments performed with actinomycin-C show more complex effects. Actinomycin initially prevents RNA synthesis and also the synthesis of enzyme-forming potential. After some minutes RNA synthesis resumes at a low rate, to be followed 4 min. later by enzyme synthesis. Enzyme-forming potential can accumulate in the presence of actinomycin after the resumption of RNA synthesis. Protein synthesis, initially inhibited by actinomycin as a consequence of the effect on RNA synthesis, is later directly inhibited by actinomycin. Adding actinomycin to de-repressed cells already making enzyme stops enzyme synthesis within 4-5 min. Enzyme synthesis resumes, as before, 4 min. after the resumption of RNA synthesis. Adding Pi together with actinomycin to de-repressed cells synthesizing enzyme does not result in a lower yield of enzyme compared with actinomycin alone. Actinomycin is less effective an inhibitor of RNA and protein synthesis in Pj-starved cells if Pi is also added. These results are discussed in view of the three main models for the regulation of enzyme induction: regulation at the level of transcription only, at translation only, or a coupled model in which transcription requires concomitant translation. It is concluded that the present evidence most powerfully supports the model of transcriptions! regulation.