MOLECULAR-CLONING AND EXPRESSION OF THE BACTERIOPHAGE T707(PROTEIN KINASE) GENE

The bacteriophage T7 0.7 gene encodes a protein which supports viral reproduction under specific suboptimal growth conditions. The 0.7 protein (gp0.7) shuts off host RNA polymerase-catalyzed transcription and also expresses a serine/threonine-specific, cAMP-independent protein kinase (PK) activity. To determine the role of the gp0.7 PK in viral reproduction, the 0.7 gene of the T7(JS78) mutant phage-whose gp0.7 expresses only the PK activity-was cloned in the plasmid expression vector pET-11 a. Cells containing the recombinant plasmid were viable, and upon IPTG induction produced a 30-kDa polypeptide, similar in size to the gp0.7-related polypeptide seen in T7(JS78)-infected cells. Extracts of cells containing this polypeptide can phosphorylate the exogenous substrate lysozyme. Expression of plasmid-encoded gp0.7(JS78) in vivo results in phosphorylation of the same proteins which are phosphorylated in T7(1S78)-infected cells; moreover, the plasmid-encoded gp0.7(JS78) is itself phosphorylated. The JS78 mutation changes GIn243 in gp0.7 to an amber codon, which explains the production of the truncated, 30-kDa gp0.7-related polypeptide, and implicates the 11-kDa C-terminal domain in host transcription shut-off. The T7(A23) 0.7 point mutant fails to express PK activity in infected cells. However, the truncated gp0.7(A23)-related polypeptide, expressed from a plasmid, exhibits PK activity in vivo and in vitro, but with an altered specificity. Thus, the A23 mutation, which changes Aspl00 to Asn, may identify a substrate recognition determinant. © 1992.