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 proheads and cuts and packages concatemeric DNA. The T4 terminase is composed of two subunits, gene products (gp) 16 and 17. The role of the small subunit, gp16, in T4 DNA packaging is not well characterized. We developed a new purification procedure to obtain large quantities of purified 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 rings, as judged by native polyacrylamide gel electrophoresis and scanning transmission electron microscopy measurements. The single ring contains 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 motif close to the N terminus of gp16 is predicted. The oligomers do not bind to DNA, but following denaturation and renaturation in the presence 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 sequence.
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Department of Biochemistry and the Molecular and Cell Biology Graduate Program, University of Maryland Medical School, Baltimore, Maryland 21201-1503, USA.
Simon
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Black
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D004247:DNA / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification*; D017348:Microscopy, Electron, Scanning Transmission; D008970:Molecular Weight; D009690:Nucleic Acid Conformation
D000345:Affinity Labels; D000255:Adenosine Triphosphate; D004247:DNA; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases