Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.
Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.
Morita
M
M
Department of Botany, Faculty of Science, Kyoto University, Japan.
Tasaka
M
M
Fujisawa
H
H
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D017903:Bacteriophage T3 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000096:biosynthesis; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D003851:Deoxyribonucleases; D000251:Adenosine Triphosphatases