Structural and functional domains of the large subunit of the bacteriophage T3 DNA packaging enzyme: importance of the C-terminal region in prohead binding.
During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19). We have developed a defined system, composed of purified gp18,gp19 and proheads for in vitro packaging of T3 DNA. Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively. These complexes associate to form a direct precursor complexes for DNA translocation into the head. The formation of the precursor complexes requires ATP as an allosteric effector. Subsequent DNA translocation is driven by ATP hydrolysis. gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis. Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-delta C10 and gp19-delta C15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions. gp19-delta C15 lacked prohead binding activity and, therefore, DNA packaging activity. gp19-delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.
Department of Botany Faculty of Science, Kyoto University, Japan.
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Morita
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Tasaka
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Fujisawa
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017903:Bacteriophage T3 / Q000201:enzymology / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017384:Sequence Deletion; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D006023:Glycoproteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D014357:Trypsin