THE CONFORMATION OF PACKAGED BACTERIOPHAGE-T7 DNA - INFORMATIVE IMAGES OF NEGATIVELY STAINED T7

Within the icosahedral protein outer shell of bacteriophage T7, a 40-k bp DNA genome occupies a cavity also occupied by a protein cylinder th at projects into the DNA from the outer shell. However, neither the in ternal cylinder nor separately resolved DNA segments are revealed in t he conventional negatively stained specimens of intact bacteriophage T 7. In the present study, a procedure of negative staining is used that reveals both internal proteins and separately resolved segments of pa ckaged DNA during electron microscopy of intact particles of a hybrid T7 bacteriophage; the hybrid is genetically T7, except for a tail fibe r gene that has a segment from the T7-related bacteriophage, T3. The n egatively stained packaged DNA segments of this hybrid bacteriophage a re found to be wrapped around the axis of the internal cylinder. To ob tain additional information about the conformation of packaged T7 DNA electron microscopy is performed of negatively stained capsids that ar e incompletely filled with DNA (ipDNA-capsids); a procedure is describ ed for improved isolation of ipDNA-capsids from lysates of hybrid bact eriophage T7-infected cells. The packaged DNA segments of ipDNA-capsid s are found not to be wrapped around any axis. Images of ipDNA-capsids are explained by the hypothesis that DNA does not achieve its wrapped condition until the capsid is more than 40% full of DNA. Wrapping via folding is, therefore, proposed to explain the images of DNA packaged in bacteriophage T7. (C) 1997 Academic Press.