In vitro packaging of bacteriophage φ29 DNA restriction fragments and the role of the terminal protein gp3

Restriction fragments of bacteriophage φ29 DNA-gp3 (DNA-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the DNA packaging protein gp16 and ATP. Both left and right end DNA-gp3 fragments were packaged in this system, in contrast to the oriented and selective packaging of left end DNA-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generally about threefold lower. In addition, certain internal (non-end) DNA fragments were packaged at efficiencies of about 10% to 15%. Digestion of the gp3 with trypsin or proteinase K reduced the packaging of whole-length DNA by a factor of 2 or 4, respectively, and removal of the gp3 from whole-length DNA or end fragments with piperidine reduced packaging to the level of internal fragments. Though the terminal protein gp3 was non-essential for DNA translocation in the defined system, it stimulated packaging of left and right end fragments, and stabilized packaging of the left end. The packaging of end and internal DNA fragments of the related phage M2Y into φ29 proheads was similar to that of φ29 DNA fragments, and certain fragments of lambda DNA were packaged at the efficiency of the internal φ29 DNA fragments. Selective packaging of DNA-gp3 left ends was restored by the addition of bacterial cell extracts or glycerol to the defined system, and these packaging conditions discriminated between φ29 and M2Y DNAs that have distinct terminal proteins.