Crellin P, Chalmers R
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
EMBO J. 2001 Jul 16;20(14):3882-91. doi: 10.1093/emboj/20.14.3882.
IHF or supercoiling is required early in Tn10 transposition, but at later stages they inhibit the reaction in a classic homeostatic loop. We investigated the mechanism of transpososome assembly and regulation using hydroxyl radical DNA protection and interference. We present a three-dimensional molecular model for the IHF-bent end of Tn10 wrapped around a transposase core. Contacts span some 80 bp at the transposon end, but after assembly of an active complex containing metal ion, most contacts become dispensable. These include transposase contacts beyond the IHF site that chaperone assembly of the complex and are needed for efficient cleavage. Single and double-end breaks do not affect the complex but divalent metal ions promote large conformational changes at bp +1 and the flanking DNA.
在Tn10转座早期需要整合宿主因子（IHF）或超螺旋，但在后期它们会在经典的稳态回路中抑制反应。我们使用羟基自由基DNA保护和干扰技术研究了转座体组装和调控的机制。我们提出了一个三维分子模型，用于描述围绕转座酶核心缠绕的Tn10的IHF弯曲末端。转座子末端的接触跨度约为80个碱基对，但在含有金属离子的活性复合物组装后，大多数接触变得可有可无。这些接触包括IHF位点以外的转座酶接触，它们陪伴复合物的组装并且是有效切割所必需的。单端和双端断裂不影响复合物，但二价金属离子会促进在碱基对+1和侧翼DNA处发生大的构象变化。
