Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage φ29 Capsids

Thorsten Hugel,Shelley Grimes,Craig L Hetherington,Dwight L Anderson,Jens Michaelis,Carlos Bustamante,Jessica M Walter,Paul J Jardine,Wayne Falk,Manfred Schliwa

doi:10.1371/journal.pbio.0050059

Abstract

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.

Highlights

As part of its viral infection cycle, the Bacillus subtilis bacteriophage u29 packages its double-stranded DNA genome into a preformed capsid shell, or prohead, by means of a powerful molecular motor [1,2]
In bacteriophage u29 assembly, the DNA passes into the shell through a channel formed by a structure called the connector
We describe a direct test of the connector rotation hypothesis, combining magnetic single-molecule manipulation techniques and single-molecule fluorescence spectroscopy

Summary

Introduction

As part of its viral infection cycle, the Bacillus subtilis bacteriophage u29 packages its double-stranded DNA genome into a preformed capsid shell, or prohead, by means of a powerful molecular motor [1,2]. The DNA-packaging motor is situated at a unique 5-fold vertex of the prohead and is a complex assembly of multiple components. At the core of the motor is the dodecameric head-tail connector, gene product 10 (gp). Associated with the connector is a ring of RNA molecules (prohead RNA or pRNA), which is required for packaging. A ring of ATPases (gp16) interacts with the pRNA to complete the packaging machinery. Gp belongs to the Her A, FtsK superfamily of ATPases [3]. Hydrolysis of ATP powers the motor and drives viral DNA into the prohead

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Results

Discussion

Conclusion