Sewitz Sven, Crellin Paul, Chalmers Ronald
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Nucleic Acids Res. 2003 Oct 15;31(20):5868-76. doi: 10.1093/nar/gkg797.
The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.
Tn10转座体在两侧具有对称的组分：有两个转座子末端，每个末端都有转座酶单体和整合宿主因子（IHF）异二聚体的结合位点。IHF的DNA弯曲活性刺激了一种中间体的组装，该中间体具有紧密折叠的转座子末端，其中转座酶具有额外的“亚末端”DNA接触，位于IHF位点的远端。这些亚末端接触是激活反应后期步骤所必需的。定量羟基自由基足迹法和凝胶阻滞展开实验表明，尽管转座体两侧具有相同的组分，但它从根本上是不对称的。转座子末端之间的主要差异定义了复合物的α侧和β侧。在没有金属离子的情况下，IHF可以从复合物β侧的转座子臂上解离。然而，IHF被锁定在复合物的α侧，可能是通过亚末端转座酶接触，直到通过金属离子依赖性构象变化释放。在反应后期，IHF抑制靶标相互作用。使用非常短的转座子臂，在饱和的IHF浓度下证明了靶标相互作用。这表明对靶标相互作用的抑制是由于单个IHF折叠的转座子臂对靶标结合位点的空间位阻。
