Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro

AbstractBacteriophage λ has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses λ procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, packaging of a mature λ genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging. When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage λ