After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [-y_32P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristies of serine phosphate: it is stable in 1 N HCl (1000) and cleaved by 1 N KOH (370) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.Bacteriophage systems have been used extensively to study the regulation of gene expression (1,2). One particular aspect of these systems is the rapidity of changes in the pattern of gene expression; phage T7 induces a whole series of control mechanisms within 4-5 min (2, 3). This rapidity of change in gene expression prompted our search for group transfers after phage infection (4-6). A phage-induced group transfer to components of the existing host machinery of gene expression would allow both a fast and an economical control.In our previous work on phage T7, we reported that labeled phosphate is incorporated into ribosomal protein after T7 infection (6,23 were then subject to SDS slab-gel electrophoresis (9); the gels were dried and placed on x-ray film overnight. Assay for Protein Kinase In Vitro. Brij-lysozyme cell extracts (10) were used as enzyme sources. The incubations were carried out at 300 for 5 min in a total volume of 0.1 ml. The incubation mixtures contained 30A]. of cell extract, 3 mg/ml of type II histone, and either (1) 15 mM sodium phosphate (pH 7.0), 0.01 MMgCl2, 0.14 mM ATP (35.7 Ci ['y32-P]ATP/mol) or (2) 25 mM Tris HCl (pH 7.0), 5 mM MgCI2, 0.1 mM EDTA, 44,AM, ATP (114 Ci of [32P]ATP/mol). The incubations were started by the addition of enzyme (cell extract) and stopped by addition of 1.5 ml of 10% trichloroacetic acid.The precipitates were boiled in trichloroacetic acid for 15 min, chilled in ice for 10 min, and collected on glass-fiber filter discs. The discs were washed 5 times with 10 ml of 10% trichloroacetic acid and once with 10 ml of methanol. After the filters were dried, radioactivity on the filters was measured by liquid scintillation spectrometry. In selected experiments, the 10% trichloroacetic acid suspensions of precipitate were heated to 900 for 20 min, cooled to ambient temperature, and centrifuged at 2500 X g. These precipit...

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Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA‐dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3′: 5′‐AMP and/or cyclic 3′: 5′‐GMP. The T7 protein kinase carries – SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7‐induced enzyme in all respects tested.