Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.

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...

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