Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli.
Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during infection of its host, Escherichia coli. The protein kinase (gp0.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphorylated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electrophoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-infected cells.
Robertson
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ES
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
Aggison
L A
LA
Nicholson
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AW
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000254:growth & development; D015180:Electrophoresis, Gel, Two-Dimensional; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007526:Isoelectric Point; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D043244:Ribonuclease III; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; C020746:factor EF-P; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli