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

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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. The life cycles of many viruses include a self-assembly stage in which a powerful molecular motor packs the DNA genome into the virus's preformed shell (the capsid). Biochemical and biophysical studies have identified essential components of the packaging machinery and measured various characteristics of the packaging process, while crystallography and electron microscopy have provided snapshots of viral structure before and after packaging. In bacteriophage φ29 assembly, the DNA passes into the shell through a channel formed by a structure called the connector. Structurally motivated models over the past 30 years have coupled DNA movement to rotation of the connector relative to the capsid. We describe a direct test of the connector rotation hypothesis, combining magnetic single-molecule manipulation techniques and single-molecule fluorescence spectroscopy. In our experiments, we use a single-dye molecule attached specifically to the connector as a reporter for its orientation and simultaneously observe the translocation of a magnetic bead attached to the DNA that is being packaged. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.