Experimental test of connector rotation during DNA packaging into bacteriophage φ29 capsids

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

doi:10.1371/journal.pbio.0050059

Experimental test of connector rotation during DNA packaging into bacteriophage φ29 capsids

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

Basic Sciences

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129 Scopus citations

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.

Original language	English (US)
Pages (from-to)	558-567
Number of pages	10
Journal	PLoS biology
Volume	5
Issue number	3
DOIs	https://doi.org/10.1371/journal.pbio.0050059
State	Published - Mar 2007

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10.1371/journal.pbio.0050059

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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.",

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AU - Hugel, Thorsten

AU - Michaelis, Jens

AU - Hetherington, Craig L.

AU - Jardine, Paul J.

AU - Grimes, Shelley

AU - Walter, Jessica M.

AU - Falk, Wayne

AU - Anderson, Dwight L.

AU - Bustamante, Carlos

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AB - 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.

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Experimental test of connector rotation during DNA packaging into bacteriophage φ29 capsids

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