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Biochemical characterization of bacteriophage lambda genome packaging in vitro
Qin Yang1, Carlos Enrique Catalano
1Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver 80262, USA.
Virology
|February 8, 2003
Summary
Bacteriophage lambda DNA packaging is inefficient in vitro. Using complex I as a substrate dramatically improves capsid utilization, revealing the energetic cost of this viral DNA packaging process.
Area of Science:
- Molecular biology
- Virology
- Biochemistry
Background:
- Bacteriophage lambda is a model system for studying viral DNA packaging.
- Limited in vitro packaging efficiency has hindered research.
- This inefficiency may stem from poor activity of purified procapsids.
Purpose of the Study:
- To improve lambda procapsid yield for better in vitro studies.
- To investigate the efficiency of different DNA substrates for packaging.
- To characterize the energetics of bacteriophage lambda DNA packaging.
Main Methods:
- Constructed a vector for high-yield lambda procapsid expression.
- Assessed in vitro packaging efficiency using mature lambda genomes.
- Evaluated packaging using concatemer DNA and complex I.
- Measured packaging-stimulated ATPase activity.
Main Results:
- Achieved a 40-fold increase in procapsid yield.
- In vitro packaging of mature genomes utilized only 4% of procapsids.
- Complex I as a substrate resulted in 40% capsid utilization.
- Identified and kinetically characterized packaging-stimulated ATPase activity.
Conclusions:
- High-yield procapsid production facilitates in vitro studies.
- Complex I is a superior substrate for efficient in vitro DNA packaging.
- Quantified the energetic cost of DNA packaging in bacteriophage lambda.