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

## TL;DR

Researchers studied how bacteriophage lambda packages DNA using purified proteins and found that a minimal system can fully replicate the process.

## Contribution

The study introduces a purified system for DNA packaging using optical tweezers and purified lambda terminase with E. coli integration host factor.

## Key findings

- Packaging events are as efficient with purified components as with crude extracts when E. coli IHF is included.
- ATP-driven DNA translocation and motor dynamics are identical in purified and extract-based systems.
- A minimal system with purified procapsids, terminase, IHF, and ATP can fully recapitulate DNA packaging.

## Abstract

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.