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Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging
1Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Journal of Molecular Biology
|March 1, 2005
Summary
Mutations in phage lambda terminase
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Large DNA viruses package DNA into preformed protein shells (proheads) using ATP hydrolysis-powered motors.
- Bacteriophages utilize terminase proteins for DNA packaging, with ATPase activity located in the large subunit's N-terminus.
- Previous studies identified lethal mutations in phage lambda gpA residues 46 and 84 affecting ATPase activity and virion assembly.
Purpose of the Study:
- To investigate the impact of conservative mutations (Y46F, Y46A) in phage lambda gpA residues 46 and 84 on DNA packaging and virion assembly.
- To elucidate the role of specific gpA residues in terminase ATPase activity and DNA translocation.
- To understand the relationship between ATPase activity, DNA packaging efficiency, and virion assembly.
Main Methods:
- Site-directed mutagenesis was used to create gpA variants (Y46F, Y46A, K84A) in phage lambda.
- In vitro assays were performed to assess terminase endonuclease, helicase, and ATPase activities.
- DNA packaging efficiency and virion assembly defects were evaluated for the mutant enzymes.
Main Results:
- Conservative gpA mutations Y46F and Y46A resulted in mild DNA packaging defects but severe post-packaging assembly defects.
- The gpA K84A mutation caused similar defects in both virion assembly and DNA packaging.
- The DNA packaging energy budget (DNA packaged/ATP hydrolyzed) remained unchanged, indicating tight coupling of translocation and ATP hydrolysis.
Conclusions:
- Phage lambda gpA residues 46 and 84 are critical for high-affinity ATPase activity, primarily influencing terminase assembly rather than translocation activity.
- The assembly of the translocation complex likely remodels the ATPase site, reducing the reliance on these specific residues for activated translocation.
- These findings provide insights into the mechanism of viral DNA packaging motors and the intricate regulation of terminase function.