Double‐stranded DNA exhibits prominent Raman bands in the spectral region 600–900 cm−1, authenticated by x‐ray crystallography as diagnostic of the macromolecular secondary structure. Since the DNA Raman fingerprint can be measured in the presence of protein, Raman spectroscopy was employed to investigate the secondary structure of dsDNA packaged within the protein coat of bacteriophage T7. Comparison of the Raman spectrum of deproteinized DNA with that of wild‐type T7 phage (52% DNA, 48% protein) reveals a packaging‐associated shift of the principal BDNA Raman marker (the band of the 825–840 cm−1 interval which is a sensitive indicator of phosphodiester geometry), without shifts of other conformation sensitive Raman markers (the bands of the 600–780 cm−1 interval which indicate sugar puckers and glycosyl torsions). The Raman marker for the protein‐free state occurs at 836 cm−1, typical of B‐form DNA of heterogeneous sequence; the marker for the packaged state occurs at 825 cm−1, characteristic of a modified B‐form DNA observed previously for specific sequences. These findings indicate different B‐form variants for dsDNA in packaged and protein‐free states. The modified B‐form secondary structure of packaged DNA is maintained when the virion is thermally burst to release the genome without removal of proteins. Comparison of the Raman spectrum of the wild‐type T7 virion with that of a deletion mutant packaging 8.4% less DNA than wild‐type (underpackaged T7), reveals the same packaging‐associated shift of the principal B‐form marker (825 cm−1) vis‐à‐vis protein‐free DNA (836 cm−1), and additional evidence of small but significant differences between phosphodiester geometries in packaged and underpackaged states. These differences are explained by a marginally (<5%) greater proportion of non‐B backbone geometry in wild‐type T7. Both wild‐type and mutant T7 exhibit Raman markers of the C2′‐endo/anti nucleoside conformations exclusively. The results are interpreted in terms of the formation of increasing amounts of non B‐form phosphodiester geometry with increasing density of DNA packaging. The data are consistent with an excess of <5% A‐type phosphodiester geometry in packaged versus underpackaged states, and 10 ± 4% excess A‐type phosphodiester geometry in packaged versus protein‐free states. Based on Raman amide I and amide III markers, the protein structures and protein‐DNA interactions in wild‐type T7 are indistinguishable from those in the T7 deletion mutant.
ASJC Scopus subject areas
- Materials Science(all)