Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine
The portal protein gp20 of bacteriophage T4 is a dodecamer located at the special five-fold vertex of the viral capsid. Its functions are essential for head assembly, genome packaging, neck and tail attachment, and genome ejection. The focus of this study was to determine the functions of the portal at the molecular level in the DNA packaging mechanism. The first challenge was determining the structure of the portal. This has been difficult because of its association with the inner E. coli membrane. It produces insoluble inclusion bodies when expressed from recombinant plasmids. To bypass this obstacle, a homology atomic model of gp20 was constructed by comparing the X-ray structures of three portals from closely related bacteriophages, namely, SPP1 gp6, phi29 gp10, and P22 gp1. These portals had a conserved structure called a "core," which was also found in the bacteriophage T4 portal. The second challenge was to find a tractable system to dissect the functions of the portal. A new biochemical approach was developed to determine the effect of mutations in various domains of the portal on function. The results show that the protruding stalk domain is required for assembly of the packaging motor; channel helices and may loops interact with DNA and assist in DNA translocation; and tunnel loops stabilize the packaged genome. A dodecamer model was also constructed and fitted into the cryoEM density of the special fivefold vertex.Together with the pentamer gp17 ATPase motor structure, the dodecamer made possible a reconstruction of the first complete phage-packaging machine. This deeper understanding of the portal structure and function could lead to new biomedical applications (e.g., translocation of genes and therapeutic molecules into cells and design of novel nano-delivery devices).
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