Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.
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.
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA. serwer@uthscsa.edu
Philip
P
Serwer
Elena T
ET
Wright
D017903:Bacteriophage T3 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D042003:DNA Packaging / Q000187:drug effects / Q000502:physiology*; D004279:DNA, Viral / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005976:Glutaral / Q000494:pharmacology; D010316:Particle Size; D012965:Sodium Chloride / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D012965:Sodium Chloride; D005976:Glutaral