Pappas Katherine M, Winans Stephen C
Department of Microbiology, 316 A Wing Hall, Cornell University, Ithaca, NY 14853, USA.
Mol Microbiol. 2003 Jul;49(2):441-55. doi: 10.1046/j.1365-2958.2003.03560.x.
The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, 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 of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. 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 regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.
根癌土壤杆菌Ti质粒的复制区域属于复制和分配系统的repABC家族，该家族成员广泛分布于α-变形菌中。最近发现，在章鱼碱型Ti质粒repABC操纵子上游区域，有三个启动子受LuxR型调节因子TraR激活。TraR对这些启动子的激活导致rep基因表达增强和Ti质粒拷贝数增加。在此，我们描述了第四个启动子，命名为P4。该启动子直接位于repA上游，不受TraR调控。通过亚克隆确定了该启动子的位置，并证明其受到强烈的自我抑制。RepA是该启动子主要的顺式作用自我抑制因子，尽管RepB增强了抑制作用，并且对于RepA介导的反式抑制作用至关重要。纯化的RepA与一个约70个核苷酸的操纵位点结合，该位点与P4启动子重叠并向下游延伸。二磷酸腺苷和三磷酸腺苷以及纯化的RepB均可增加结合亲和力。P1、P2和P3启动子的激活增强了P4的活性，这表明P4以某种方式与上游启动子相互作用。这些发现表明，自诱导和自我抑制在调节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且相反的作用。
