| Research Abstract |
During head assembly of double-stranded DNA phages, phage DNA is selected and translocated into a preformed protein shell, called the prohead, by hydrolysis of ATP.The molecular mechanisms of the selective packaging are specific for each phages and the energetic mechanism of the DNA translocation is common among phages.
T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of noncapsid proteins, gp18 and gp19. We showed that recombinant plasmids carrying DNA sequences necessary for DNA packaging(Pac sequences)can be packaged into transducing particles. The pac sequence has a bipartite structure consisting of the cleavage sites for processing of concatemeric DNA and the sequence containing a promoter sequence for T3 RNA polymerase. Promoter activity is necessary for DNA packaging and the transcriptional specificity determi
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nes the DNA packaging specificity of T3 and T7.
We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, in which DNA is packaged by a way of a concatemer as an intermediate, for in vitro packaging of T3 DNA.The defined system displays an ATPase activity, composed of DNA packaging-dependent and -independent ATPases. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.gp19 has ATP binding activity and three ATP binfding and two Mg^<2+> binding consensus motifs in its amino acid sequence. We have studied the roles of gp19 in the DNA packaging reaction by molecular genetic analysis by site-directed mutagenesis or deletion mutations. The results indicate that gp19 plays crucial roles in DNA translocation and in procssing of concatemric DNA.gp19 undergoes conformational changes in multiple steps of DNA packaging process through interaction with ATP.We have proposed a model for DNA translocation in which the gp19-connector located at the portal vertex would translocate DNA into the prohead by a cyclic change in the conformation of gp19 mediated by ATP. Less
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