Single-molecule measurements of bacteriophage lambda DNA packaging using purified terminase motor proteins and E. coli integration host factor.

Biomotor-driven DNA packaging is a key step in the life cycle of many viruses. We previously developed single-molecule methods using optical tweezers to measure packaging dynamics of the bacteriophage lambda motor. The lambda system is more complex than others examined via single-molecule assays with respect to the packaging substrate and ancillary proteins required. Because of this, previous studies which efficiently detected packaging events used crude E. coli cell extracts containing host factors and the terminase packaging enzyme. However, use of extracts is suboptimal for biochemical manipulation and obfuscates interrogation of additional factors that affect the process. Here we describe an optical tweezers assay using purified lambda terminase holoenzyme. Packaging events are as efficient as with crude extracts, but only if purified E. coli integration host factor (IHF) is included in the motor assembly reactions. We find that the ATP-driven DNA translocation dynamics, motor force generation, and motor-DNA interactions without nucleotide are virtually identical to those measured with extracts. Thus, single-molecule packaging activity can be fully recapitulated in a minimal system containing only purified lambda procapsids, purified terminase, IHF, and ATP. This sets the stage for single-molecule studies to investigate additional phage proteins known to play essential roles in the packaging reaction.