Single Molecule- and Fluorescence Correlation Spectroscopy-FRET Analysis of Phage DNA Packaging: Co-localization of the Packaged Phage T4 DNA Ends within the Capsid

摘要

Linear DNAs of any sequence can be packaged with high efficiency in vitro into empty viral procapsids by the phage T4 terminase. Packaging substrates of 5 and 50 kbps, terminated by energy transfer dye pairs, were constructed from plasmid and lambda phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ~20% of the substrate DNA was packaged and the DNA dye ends of the packaged DNA were protected from nuclease digestion. Both 5 and 50 kbp DNAs upon packaging produced comparable FRET (fluorescence resonance energy transfer) between the Cy5 and Cy5.5 two-dye terminated DNAs. Single molecule FRET (smFRET) and photobleaching analysis shows that the FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single molecule detection (SMD) allows mechanistic analysis of packaging in vitro. FCS- and sm-FRET measurements are comparable, and show that both the 5 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.