Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4.

Reza Vafabakhsh,Li Dai,Kyung Suk Lee,Karin A Dahmen,Venigalla B Rao,Zhihong Zhang,Tyler Earnest,Kiran Kondabagil,Taekjip Ha

doi:10.1073/pnas.1407235111

Abstract

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.

Highlights

Viral DNA packaging motors are among the most powerful molecular motors known
We report a single-molecule fluorescence assay that allowed us to dissect packaging initiation starting from a dsDNA end, in real time, by the phage T4 DNA packaging machine
DNA packaging into a viral capsid is a complex process consisting of initiation, elongation, and termination

Summary

Introduction

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and singlemolecule biophysical approaches have been used to understand their mechanochemistry. In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large “terminase” proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end [7, 8]. The large terminase, which is an ATPase, attaches to the protruding end of the portal and assembles into an oligomeric motor that translocates the DNA genome into the empty prohead through the ∼3.5-nmdiameter portal channel using energy from ATP hydrolysis [7, 8]. An electrostatic forcedriven translocation mechanism was proposed in which gp subunits alternate between the “tensed” (compact) and “relaxed” (extended) conformational states that is coupled to translocation of DNA in a piston-like fashion [12]

Methods

Results

Conclusion