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Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate
Author(s) -
Serwer Philip,
Wright Elena T.
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100326
Subject(s) - agarose gel electrophoresis , capsid , dna , agarose , biophysics , chemistry , in vivo , cleavage (geology) , gel electrophoresis , microbiology and biotechnology , crystallography , biology , biochemistry , genetics , gene , paleontology , fracture (geology)
We find a new aspect of DNA packaging‐associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross‐linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two‐dimensional agarose gel electrophoresis (2D‐AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA‐capsids. We initially increase the production of ipDNA‐capsids by raising NaCl concentration during in vivo DNA packaging. By 2D‐AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA‐capsid. The contracted shell‐state is found when the ipDNA length/mature DNA length ( F ) is above 0.17, but not at lower F . Some contracted‐shell ipDNA‐capsids have the phage tail; others do not. The contracted‐shell ipDNA‐capsids are explained by premature DNA maturation cleavage that makes accessible a contracted‐shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA‐capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging.