Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
Electrophoresis. 2012 Jan;33(2):352-65. doi: 10.1002/elps.201100326.
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.
我们发现噬菌体 T3 衣壳与 DNA 包装相关的结构流变性的一个新方面。该方法是：（i）用戊二醛交联体内 DNA 包装中间体以稳定结构，然后（ii）通过二维琼脂糖凝胶电泳（2D-AGE）确定有效半径。中间体是不完全包装 DNA（ipDNA）的衣壳和没有外部 DNA 片段的衣壳；这些中间体称为 ipDNA 衣壳。我们最初通过在体内 DNA 包装过程中提高 NaCl 浓度来增加 ipDNA 衣壳的产量。通过 2D-AGE，我们发现了一种以前鉴定的 ipDNA 衣壳的一些颗粒的新的收缩壳状态。当 ipDNA 长度/成熟 DNA 长度（F）大于 0.17 时，会出现收缩壳状态，但在较低的 F 时不会出现。一些收缩壳 ipDNA 衣壳带有噬菌体尾部，而另一些则没有。收缩壳 ipDNA 衣壳是由 DNA 过早成熟切割引起的，这使得 T3 DNA 包装马达循环的收缩壳中间体变得可用。对 ipDNA 衣壳的分析，而不是对未切割 DNA 的中间体的分析，为体内 DNA 包装的完整生化分析提供了一种简化策略。
