PURIFICATION AND CHARACTERIZATION OF THE SMALL-SUBUNIT OF PHAGE-T4 TERMINASE, GP16, REQUIRED FOR DNA PACKAGING

Phage T4 terminase is an enzyme that binds to the portal protein of pr oheads and cuts and packages concatemeric DNA. The T4 terminase is com posed of two subunits, gene products (gp) 16 and 17. The role of the s mall subunit, gp16, in T4 DNA packaging is not well characterized, We developed a new purification procedure to obtain large quantities of p urified gp16 from an overexpression vector. The pure protein is found in two molecular weight forms, due to specific C-terminal truncation, displays in vitro packaging activity, and binds but does not hydrolyze ATP, gp16 forms specific oligomers, rings, and side-by-side double ri ngs, as judged by native polyacrylamide gel electrophoresis and scanni ng transmission electron microscopy measurements. The single ring cont ains about eight monomers, and the rings have a diameter of about 8 nm with a central hole of about 2 nm. A DNA-binding helix-turn-helix mot if close to the N terminus of gp16 is predicted. The oligomers do not bind to DNA, but following denaturation and renaturation in the presen ce of DNA, binding can be demonstrated by gel shift and filter binding assays, gp16 binds to double-stranded DNA but not single-stranded DNA , and appears to bind preferentially to a gene 16-containing DNA seque nce.