Regulation of the synthesis of phage-induced proteins in Escherichia coli B

Abstract

The control of protein synthesis in protoplasts of bacteriophage T4-infected E. coli has been studied in a two part incubation in which the processes of transcription and translation have been separated by the use of inhibitors of RNA and protein synthesis. The accumulation of specific messenger ribonucleic acids during the first incubation in the presence of an inhibitor of protein synthesis (5-methyltryptophan or 5-methyltryptophan plus chloramphenicol) was analyzed by preventing further transcription with rifamycin B, and following the formation of early enzymes in the second period upon restoration of protein synthesis. From the results of these studies the early enzymes induced by infection with T4 could be classified into at least three groups, distinguished operationally by the conditions required for the transcription of the respective messengers. (a) The messenger for thymidylate kinase could not be detected until protein synthesis was restored, an indication that a phage-induced protein is necessary for the transcription of this region of the phage genome. (b) The messenges for dCTPase, dCMP hydroxymethylase, thymidylate synthetase, and dihydrofolate reductase were synthesized during incubation with 5-methyltryptophan. In the case of dCTPase and dCMP hydroxymethylase, the amount of messenger that accumulates in the presence of 5-methyltryptophan is sufficient to direct the formation of the normal level of these enzymes when protein synthesis is restored with tryptophan. Normal kinetics of synthesis of the thymidylate synthetase were only observed when transcription was allowed to occur with concomitant protein synthesis. The synthesis of dihydrofolate reductase, on the other hand, was not inhibited by 5-methyltryptophan. (c) In the presence of chloramphenicol, under conditions in which protein synthesis was completely inhibited, the messengers for dihydrofolate reductase and dCMP hydroxymethylase accumulated, as determined by their ability to direct the formation of these enzymes when protein synthesis was restored. The synthesis of messengers for dCTPase, thymidylate synthetase and thymidylate kinase did not accumulate during incubation with chloramphenicol. By use of the technique of DNA-RNA hybridization it was demonstrated that a selective group of early messengers are synthesized in the presence of chloramphenicol, in agreement with the above studies of enzyme synthesis. These observations suggest that the transcription of the early region of the phage genome may be regulated by protein synthesis. It remains to be determined whether specific phage-induced proteins are required for transcription at certain sites on the phage DNA, or whether there is a non-specific requirement for concomitant translation. The relationship of DNA replication to the control of early enzyme synthesis and the formation of a late protein, the phage tail-fiber, was studied in cells infected with a mutant, T4 am130. The onset and the extent of DNA synthesis were regulated in these cells by use of fluorodeoxyuridine and thymidine. It was found that DNA replication is necessary for the transcription of the late region of the phage genome, and thus for the synthesis of the late proteins. In addition, the sustained synthesis of late RNA and the tail-fiber was shown to require the continued replication of DNA. The mechanism regulating the cessation of early enzyme synthesis appears to become operative shortly after the onset of DNA replication.