DNA packaging motor assembly intermediate of bacteriophage phi29

Area of Science:

• Molecular Biology
• Virology
• Structural Biology

Background:

• Understanding DNA packaging in tailed double-stranded DNA bacteriophages is key to DNA translocation mechanisms.
• Bacteriophage phi29's packaging ATPase, gene product 16 (gp16), plays a critical role in this process.

Purpose of the Study:

• To investigate the structure and assembly of bacteriophage phi29's packaging ATPase (gp16).
• To elucidate the mechanism of packaging motor formation and its role in DNA translocation.

Main Methods:

• Overexpression and purification of gp16 in Bacillus subtilis.
• Assembly of gp16 with phi29 precursor capsid (prohead) and prohead RNA (pRNA).
• Characterization using analytical ultracentrifugation, gel filtration, electron microscopy, and cryo-electron microscopy.

Main Results:

• gp16 forms higher-order oligomers concentration-dependently.
• gp16 binding to the prohead requires an oligomeric pRNA complex.
• gp16 specifically binds the A-helix of pRNA, protecting specific residues.
• Cryo-EM revealed the complete packaging motor complex structure.
• The complex is active in DNA packaging and supercoiling.

Conclusions:

• gp16 assembly onto the prohead/pRNA complex forms an active DNA packaging motor.
• This assembly pathway represents an alternative to initial binding to DNA.
• Findings provide insights into the mechanism of DNA translocation in bacteriophages.