Cooperative DNA Binding of the Plasmid Partitioning Protein TubR from the Bacillus cereus pXO1 Plasmid
Cooperative DNA Binding of the Plasmid Partitioning Protein TubR from the Bacillus cereus pXO1 Plasmid
复制标题
蜡状芽孢杆菌 pXO1 质粒中质粒分配蛋白 TubR 的协同 DNA 结合
DOI：
10.1016/j.jmb.2018.11.001
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
S. Fuchigami
中科院分区：
文献类型：
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作者：
I. Hayashi;T. Oda;M. Sato;S. Fuchigami
Tubulin/FtsZ样GTubZ负责维持毒力芽孢杆菌中pXO 1样质粒的稳定性，TubZ以GTP依赖的方式形成丝状体，并且像其他低拷贝质粒的分配系统一样，它需要连接质粒和TubZ丝状体的着丝粒结合蛋白TubR。调节TubZ分配的系统已经在梭菌原噬菌体以及芽孢杆菌中被鉴定，其中TubZ通过结合和牵引分离体（segrosome）来促进分配：由TubR和着丝粒组成的核蛋白复合物。然而，对segrosome组装的分子机制以及segrosome与TubZ丝之间的瞬时开关相互作用仍然知之甚少。在这里，我们确定了TubR从蜡状芽孢杆菌的晶体结构在2.0的分辨率和研究的DNA结合能力的TubR使用羟基自由基足迹和电泳迁移率变动分析。TubR二聚体具有2倍对称性，并结合到tubRZ启动子区的15-bp回文共有序列。连续的TubR结合位点彼此重叠，这使得TubR能够以协同方式有效结合。有趣的是，segrosome采用了扩展的DNA-蛋白质丝结构，并可能通过以不对称的方式将非共有残基引入回文中来获得构象灵活性。总之，我们的实验结果和结构模型表明，独特的着丝粒识别机制的TubR允许瞬时复合物之间的segrosome和动态聚合物的TubZ的形成。
Tubulin/FtsZ-like GTPase TubZ is responsible for maintaining the stability of pXO1-like plasmids in virulent Bacilli.TubZ forms a filament in a GTP-dependent manner, and like other partitioning systems of low-copy-number plasmids, it requires the centromere-binding protein TubR that connects the plasmid to the TubZ filament. Systems regulating TubZ partitioning have been identified in Clostridium prophages as well as virulentBacillusspecies, in which TubZ facilitates partitioning by binding and towing the segrosome: the nucleoprotein complex composed of TubR and the centromere. However, the molecular mechanisms of segrosome assembly and the transient on–off interactions between the segrosome and the TubZ filament remain poorly understood. Here, we determined the crystal structure of TubR from Bacillus cereus at 2.0-Å resolution and investigated the DNA-binding ability of TubR using hydroxyl radical footprinting and electrophoretic mobility shift assays. The TubR dimer possesses 2-fold symmetry and binds to a 15-bp palindromic consensus sequence in thetubRZpromoter region. Continuous TubR-binding sites overlap each other, which enables efficient binding of TubR in a cooperative manner. Interestingly, the segrosome adopts an extended DNA–protein filament structure and likely gains conformational flexibility by introducing non-consensus residues into the palindromes in an asymmetric manner. Together, our experimental results and structural model indicate that the unique centromere recognition mechanism of TubR allows transient complex formation between the segrosome and the dynamic polymer of TubZ.