The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon.
The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon.
复制标题
RepA 和 RepB 自阻遏物以及 TraR 在 Ti 质粒 repABC 操纵子的调节中发挥相反的作用。
DOI：
10.1046/j.1365-2958.2003.03560.x
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发表时间：
2003
影响因子：
3.6
通讯作者：
Winans,StephenC
中科院分区：
文献类型：
--
作者：
Pappas,KatherineM;Winans,StephenC
根癌农杆菌Ti质粒的复制区属于复制和分配系统repABC家族，其成员广泛分布于α变形菌中。在章鱼碱型 Ti 质粒repABCoperon 的上游区域，最近显示三个启动子被 LuxR 型调节因子 TraR 激活。 TraR 激活这些启动子导致rep基因表达增强并增加Ti质粒拷贝数。在这里，我们描述了第四个启动子，指定为 P4。该启动子位于repA的直接上游并且不受TraR调节。启动子通过亚克隆定位并被证明具有强烈的自抑制作用。 RepA 是该启动子的主要顺式作用自抑制因子，尽管 RepB 增强了抑制作用，并且对于 RepA 介导的反式抑制至关重要。纯化的 RepA 与大约 70 个核苷酸的操纵位点结合，与 P4 启动子重叠并充分延伸到下游。二磷酸腺苷和三磷酸腺苷以及纯化的 RepB 可以增加结合亲和力。 P1、P2 和 P3 的激活增强了 P4 的活性，表明 P4 以某种方式与上游启动子进行通信。这些发现表明，自诱导和自抑制在调节repABC表达以及Ti质粒的复制、稳定性和拷贝数方面发挥着关键和相反的作用。
The replicator regions of the Ti plasmids ofAgrobacterium tumefaciensbelong to therepABCfamily of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine‐type Ti plasmidrepABCoperon, three promoters were recently shown to be activated by the LuxR‐type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream ofrepAand is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the majorcis‐acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA‐mediated repression intrans. Purified RepA bound to an approximately 70‐nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di‐ and tri‐phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulatingrepABCexpression and hence in the replication, stability and copy number of the Ti plasmid.