Isolation and characterization of T4 bacteriophage gp17 terminase, a large subunit multimer with enhanced ATPase activity.
Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional column steps completed purification. The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging. Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.
Baumann
Richard G
RG
Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201-1503, USA.
Black
Lindsay W
LW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000502:physiology; D004706:Endodeoxyribonucleases / Q000032:analysis / Q000302:isolation & purification; D011487:Protein Conformation; D021122:Protein Subunits / Q000032:analysis / Q000302:isolation & purification; D014764:Viral Proteins / Q000032:analysis / Q000302:isolation & purification; D019065:Virus Assembly
D021122:Protein Subunits; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases