Molecular cloning and expression of the bacteriophage T7 0.7(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-11a. 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(JS78)-infected cells; moreover, the plasmid-encoded gp0.7(JS78) is itself phosphorylated. The JS78 mutation changes Gln243 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 T7(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 Asp100 to Asn, may identify a substrate recognition determinant.
Michalewicz
J
J
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
Nicholson
A W
AW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011995:Recombination, Genetic; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D006023:Glycoproteins; D014764:Viral Proteins; D010770:Phosphotransferases; D011494:Protein Kinases; C072445:0.7 protein, Enterobacteria phage T7; D017346:Protein Serine-Threonine Kinases