Isolation and Characterization of T4 Bacteriophage gp17 Terminase, a Large Subunit Multimer with Enhanced ATPase Activity

Abstract

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 (ϳ10% 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 (ϳ400 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. Many dsDNA1 bacteriophage require the activity of two "terminase" proteins to cut and package concatameric DNA into assembled phage proheads. In T4, the gp16 (small subunit) and gp17 (large subunit) terminase proteins are essential proteins for DNA packaging. (Similar proteins are gpA and gpNu1 in phage, gp18 and gp19 in phage T7/T3, gp2 and gp3 in phage P22, and gp3 and gp16 in phage 29.) As generally found among these phage terminases, the T4 small subunit confers the specific DNA-binding/association properties (1-3). The large subunit contains the prohead-binding and putative DNAtranslocating ATPase activities (3-7). However, because the measured in vitro ATPase activities of isolated terminase proteins (8, 9) have been low in comparison with measured rates of ATP consumption in packaging (10, 11), the identification of terminases as DNA-translocating ATPases has been problematic. What is clear is that packaging is ATP-driven and involves the cooperation of the DNA-associated terminase proteins with the prohead-associated portal vertex protein (7, 12, 13) in phage T4, gp20.The T4 terminase large subunit protein, gp17, has proven difficult to purify because of the protein's limited solubility and toxicity to host cells (14). Coexpression with the gp16 small subunit reduces toxicity; thus, gp17 could be purified with gp16 in low amounts from a temperature-induced expression vector (4). A vector that makes gp17 with a 20-amino acid OmpT leader peptide results in low amounts of uninduced, soluble, and active protein, whereas the overexpressed protein is insoluble and inactive and resists renaturation (15). A His-tagged gp17 protein was also overexpressed and showed similar problems in protein solubi...

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

“…S6). This alternative model may not necessarily be incompatible with previous data suggesting interactions between portals and terminase large subunits, because: (i) the terminase large subunit gp17 in T4 showed a multidomain architecture with extended linker regions among domains (7) which can potentially allow conformational change needed to reach the portal, and (ii) terminase small subunits stimulate the ATPase activity of large subunits, an activity required for DNA-packaging, as shown in phages T4 (34,35), SPP1 (36) and lambda (37), and this stimulation may result from conformational change of large subunits induced by binding of small subunits (38). A feature of this alternative model is that both terminase subunits participate in molecular interactions with the portal.…”

Functional Analysis of the Bacteriophage T4 DNA-packaging ATPase Motor

“…Because a number of components constitute the packaging machine, and several ATPase sites have been identified (10,(21)(22)(23)39), the ATPase that powers DNA translocation remained uncertain. We have earlier demonstrated that the T4 large terminase protein gp17 exhibits ATPase and in vitro DNA packaging activities that are stimulated 50 -100-fold by the small terminase protein gp16 (11)(12)(13)40). Sequence alignments mapped a key ATPase in the N-terminal half of gp17 (10,14,15).…”

“…Molecular genetics and biochemical evidence implicate this ATPase in DNA packaging. The evidence includes the following: (i) the T4 gp17 alone exhibits ATPase and in vitro DNA packaging activities (11,12); (ii) both the activities are stimulated 50 -100-fold by the small terminase protein gp16 (12,13); (iii) the N-terminal Walker A motif, 161 SRQLGKT 167 , is critical for function and any substitution in the conserved residues results in a loss of stimulated ATPase and in vitro DNA packaging activities (14); and (iv) a critical catalytic carboxylate, Glu 256 , which is involved in the cleavage of the ␤,␥-phosphoanhydride bond of ATP (15), has been identified.…”

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

“…The ac and acq gp 17 genes were cloned as the wt gp 17 gene in pTYB2 plasmids and were expressed in BL21 (DE3) pLysS, successfully making the gp17-intein/chitinbinding domain fusion (27). Full-length active ac and acq gp17 was purified based on the Impact system of cloning (New England Biolabs, Inc.).…”

“…To make sure the sample was equilibrated-that is, that it only gave one band in electrophoresis-the samples were always run on 1.5% (wt/vol) agarose and at a 100 V constant field electrophoresis. Western blotting was carried out using a gp17 antiserum as previously described (27) after running the samples on 5% native PAGE gel under cold conditions.…”