INHIBITION OF DEOXYRIBONUCLEIC ACID-DIRECTED RIBONUCLEIC ACID POLYMERASE IN ESCHERICHIA COLI AFTER INFECTION WITH BACTERIOPHAGE T4.

Astrachan and Volkin' have observed that a metabolically labile form of RNA2 with a base composition comparable to that of the invading phage DNA is formed early in phage infection. At least from a quantitative point of view, the turnover of this RNA (messenger RNA)3-5 decreases with increasing time after infection.1 According to current concepts,3-5 the production of messenger RNA and hence phage-specific proteins should be directly related to the activity of DNA-directed RNA polymerase.6-'0 One such group of phage-specific proteins are the "early enzymes,"11 which are related to the formation and incorporation of 5-hydroxymethyl deoxycytidylatel2 into phage DNA. These enzymes appear shortly after phage infection but their production abruptly ceases at about 12-15 min.13 14 We have found that under the conditions of our assay the level of the activity of DNA-directed RNA polymerase in E. coli B decreases markedly during the first 10-12 min after infection with T4 bacteriophage. A relatively heat-stable substance is produced that can effectively inhibit purified RNA polymerase of uninfected cells. The production of the inhibitor is arrested by the addition of chloramphenicol to cells shortly after their infection with bacteriophage. In a corresponding although not similar situation Franklin and Baltimore15 have previously reported the lowering of the DNA-directed RNA polymerase activity of L-cell nuclei following infection with the RNA-containing mengovirus. Materials and Methods. -The bacteriophage T4 and its mutant am N8216 17 were grown in E. coli B and E. coli CR 63, respectively. After complete lysis the debris was removed by centrifugation for 30 min at 5000 X g. The bacteriophage were sedimented by centrifugation at 20,000 X g for one hour at 0WC; the pellets were covered with buffer solution (0.001 M potassium phosphate, pH 7.0, 0.001 M MgCl2, gelatin, 100 mg/l) for several days and finally suapendea by stirring. Bacteriophage DNA was prepared by phenol extraction as described by Davisoirand Freifelder. '8 19 C14-labeled ATP and CTP were purchased from Schwarz BioResearch, Inc. and the unlabeled nucleoside triphosphates, ATP, CTP, GTP, and UTP from Pabst Labqratoris'; Milwaukee, Wisconsin. The preparation of C14-labeled dHMP has been described.47 Chloramphenicol and streptomycin sulfate were obtained from Parke, Davis and Company and E. R. Squibb and Sons, respectively. Sephadex G-25 was obtained from Pharmnacia, Uppsala, Sweden. Preparation of extracts from infected bacteria: Cells of E. coli B were grown in the medium of Fraser and Jerrel2' to a concentration of 109 per ml and were then infected with T4 or T4 am N82 at the desired multiplicity. In some instances 4 phage per bacterium were added, and 4 min later, the same amount of phage was added again, to give a final multiplicity of 8. At various intervals after infection, a 400-ml aliquot of cell suspension, containing about 5 gm of cells, was siphoned off into 400 ml of frozen, crushed Fraser and Jerrel medium, and was then centrifuged at 5000 X g for 40 min in a refrigerated Stock centrifuge. The packed cells from each sample were frozen after the addition of 5 ml of buffer A (0.01 M Tris buffer, pH 7.9, 0.01 M MgCl2, and 0.0001 M EDTA), and then ruptured in a Hughes press.22 The pressed material was weighed, thawed, and centrifuged at about 20,000 X g for 30 min at 00C. The ribosomes were then sedimented by further centrifugation for 120 min at 34,000 rpm in the size 40 head of the model L Spinco preparative centrifuge, and the supernatant fluid was carefully removed. A 14.3 M solution of 2-