Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and lambda phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that approximately 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.
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