Sa. Overman et al., CONFORMATION AND INTERACTIONS OF THE PACKAGED DOUBLE-STRANDED DNA GENOME OF BACTERIOPHAGE-T7, Biospectroscopy, 4(5), 1998, pp. 47-56
Citations number
33
Categorie Soggetti
Biochemical Research Methods",Spectroscopy,Biophysics
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.