Tang Y, Lichtenstein C, Cotterill S
Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, UK.
Nucleic Acids Res. 1991 Jun 25;19(12):3395-402. doi: 10.1093/nar/19.12.3395.
Tn7, a large bacterial transposon encodes 5 proteins required for its transposition. We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain. This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration. DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected. Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein. ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins. Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix.
Tn7是一种大型细菌转座子，编码其转座所需的5种蛋白质。我们报告了一种从过表达菌株中快速简便地纯化其中一种蛋白质TnsB的方法。在凝胶迁移实验中，该蛋白质被证明以两个不同阶段结合到Tn7的末端，这是蛋白质浓度的函数。在Tn7两端进行的DNA酶足迹实验表明，TnsB识别序列（一组22 bp重复序列）加上Tn7末端受到保护。TnsB的结合似乎具有协同性，但仅在蛋白质的阈值浓度以上才能观察到。ATP和Mg2+对保护模式没有影响，添加其他Tn7编码的蛋白质也没有影响。在右端进行的羟基自由基足迹实验表明，TnsB优先结合到DNA螺旋的一侧。
