A new procedure for the purification of the bacteriophage lambda terminase enzyme and its subunits. Properties of gene product A, the large subunit.
New methods for the purification of highly active bacteriophage lambda terminase holoenzyme, and its individual subunits, gene products (gp) A and gpNu1, have been developed. These methods are rapid, simple, reproducible, and give high yields of unaggregated protein from small volumes of culture. The procedures involve fractionation of extracts of Escherichia coli strains harboring plasmids engineered to overproduce the respective proteins. All purified proteins exist as monomers or dimers at moderate concentrations. At concentrations where holoenzyme efficiently promotes in vitro cosN-cleavage and lambda DNA packaging, gpA displays neither of these activities unless supplemented with gpNu1 and the E. coli protein integration host factor. At high protein concentrations, however, gpA can promote cos-cleavage by itself. Although gpNu1 itself cannot promote either cosN-cleavage or DNA packaging, it does modulate these activities of gpA. GpA is a DNA-stimulated ATPase whose catalytic parameters closely resemble those of the holoenzyme. Like the holoenzyme, gpA displays a DNA helicase activity which is able to melt the annealed cosN overhangs. Certain preparations of gpA appear to undergo a time-dependent amino-terminal clipping at discrete sites even in the presence of as many as four protease inhibitors and at low temperature.
Parris
W
W
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Rubinchik
S
S
Yang
Y C
YC
Gold
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D010582:Bacteriophage lambda / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral
D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases