Conformational toggling controls target site choice for the heteromeric transposase element Tn7.
Conformational toggling controls target site choice for the heteromeric transposase element Tn7.
复制标题
构象切换控制异聚转座酶元件 Tn7 的靶位点选择。
DOI：
10.1093/nar/gkv913
复制
发表时间：
2015
影响因子：
14.9
通讯作者：
Peters,JosephE
中科院分区：
文献类型：
--
作者：
Shi,Qiaojuan;Straus,MarcoR;Caron,JeremyJ;Wang,Huasheng;Chung,YuSeon;Guarné,Alba;Peters,JosephE
细菌转座子 Tn7 通过使用元件编码蛋白 TnsE 引导转座到活跃复制的 DNA 中，从而促进水平转移。对野生型 TnsE C 端结构域的结构分析发现了一种新的蛋白质折叠，其中包括一个在两种不同构象之间切换的中央 V 形环。强大的 TnsE 活性增益变体的结构将该环锁定在单个构象中，表明构象灵活性调节 TnsE 活性。对一系列 TnsE 突变体进行基于结构的分析，将转座活性与 DNA 结合稳定性联系起来。野生型 TnsE 似乎与靶标 DNA 自然形成不稳定的复合物，而转座频率和靶向发生较大变化所需的突变体组合稳定了这种相互作用。总的来说，我们的工作揭示了一种独特的结构校对机制，其中两种构象之间的切换通过限制目标 DNA 接合的稳定性来调节目标承诺，直到确定适当的插入位点。
The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified.