Purification and characterization of T7 head-tail connectors expressed from the cloned gene.
Cloned gene 8, which specifies the protein of the head-tail connector of bacteriophage T7, was expressed in Escherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography perhaps because of dissociation of monomer-connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.
Cerritelli
M E
ME
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Studier
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FW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics*; D003001:Cloning, Molecular; D004270:DNA, Circular; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D005814:Genes, Viral; D017348:Microscopy, Electron, Scanning Transmission; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure
D004270:DNA, Circular; D014764:Viral Proteins; D000251:Adenosine Triphosphatases