CONFORMATION AND INTERACTIONS OF THE PACKAGED DOUBLE-STRANDED DNA GENOME OF BACTERIOPHAGE-T7

The structure of the packaged double-stranded DNA genome of bacterioph age T7 was compared to that of unpackaged T7 DNA using digital differe nce Raman spectroscopy. Spectral data were obtained at 25 degrees C fr om native T7 virus (100 mg/mL), empty T7 capsids (50 mg/mL), and purif ied T7 DNA (40 mg/mL) in buffer containing 200 mM NaCl, 10 mM MgCl2, a nd 10 mM Tris at pH 7.5. At these conditions, the local conformation o f T7 DNA was not affected by packaging. Specifically, the local B-form secondary structure of unpackaged T7 DNA, including furanose C2'-endo pucker, anti glycosyl torsion, Watson-Crick base pairing, and base st acking, were essentially fully (>98%) retained when the genome was con densed within the viral capsid. However, the average electrostatic env ironment of T7 DNA phosphates was altered dramatically by packaging as revealed by large perturbations in the Raman bands associated with lo calized vibrations of the DNA phosphate groups. The change in the phos phate environment was attributed to Mg2+ ions that were packaged with the genomic DNA, and the observed Raman perturbations of genomic DNA w ere equivalent to those generated by a 50-100-fold increase in Mg2+ co ncentration in aqueous phosphodiester model compounds. The T7 data wer e qualitatively and quantitatively similar to those observed previousl y for packaged DNA of bacteriophage P22 and imply that genomic DNAs of T7 and P22 are both organized in a similar fashion within their respe ctive capsids. The results show that the condensed genome does not con tain kinks or folds that would disrupt the local B conformation by mor e than 2%. The present findings are discussed in relation to previousl y proposed models for condensation and organization of double-stranded and single-stranded viral DNA. (C) 1998 John Wiley & Sons, Inc.