Cryo-EM analysis of DNA packaging by the HK97 bacteriophage

DNA packaging is a key step in the assembly of dsDNA viruses, such as tailed bacteriophages and evolutionarily related herpes viruses, whereby empty capsids are filled with a copy of the viral genome. The task is mediated by an ATP powered DNA packaging motor, made of terminase proteins, interacting with the portal vertex of a capsid. Cos phages use a defined signal, recognised by the terminase machinery, to mark the beginning and end of their genome in newly replicated, concatemeric DNA. This thesis focuses on cryo-EM analysis of the DNA packaging machine from the Escherichia coli cos bacteriophage HK97. A monomeric crystal structure of large terminase (PDB 6Z6D) is available, but accurate structural information for assemblies that initiate, process, and terminate packaging remained unknown. The motor was assembled in vitro, using proheads and terminase proteins purified separately, and DNA protection assays indicated that low concentrations of ATP could stall the packaging motor at a cos site of the substrate DNA. This behaviour permitted particles with an assembled packaging motor to be selected using a novel magnetic bead assay, that were suitable for structural studies. Cryo-EM reconstructions elucidated a high-resolution structure of prohead II and the portal protein. An in situ asymmetric reconstruction of the portal protein was also achieved, showing unique contacts between the portal and surrounding capsomeres. Reconstruction of the motor displayed a ring of 5 large terminase monomers with DNA in the central channel. The motor is tilted relative to the portal axis, with only a single C-terminal domain of the terminase making clear contact. The N-terminal ATPase domains, constitute a second ring below, displaying variable degrees of extension away from the C-terminal domains. This is indicative of a novel packaging mechanism, where contraction between the 2 large terminase domains appears to drive translocation, with terminase-portal interactions varying throughout the ATP hydrolysis cycle.