Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control
Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control
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DOI：
10.1111/j.1365-2958.2009.06755.x
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发表时间：
2009-08-01
影响因子：
3.6
通讯作者：
Winans, Stephen C.
中科院分区：
文献类型：
--
作者：
Costa, Esther D.;Cho, Hongbaek;Winans, Stephen C.
根癌农杆菌 Ti 质粒复制和接合转移所需的基因受到群体感应转录因子 TraR 的调节，其 N 端结构域与信息素 3-氧代-辛酰高丝氨酸内酯 (OOHL) 结合，其 C 端结构域与称为 tra 盒的特定 DNA 序列结合。在这里，我们构建了 117 个突变体，改变了 TraR N 端结构域的 103 个表面暴露的氨基酸残基。测试每个突变体的traI启动子和traM启动子的激活，其中TraR与转录起始位点上游以45个核苷酸为中心的位点结合，其中TraR结合以上游66个核苷酸为中心的位点。 18 个残基的改变阻断了 traI 启动子的活性。其中，三个位置的改变损害了 TraR 丰度或 DNA 结合，留下了阳性对照特别需要的 15 个残基。在这 15 个残基中，有 9 个也阻断或降低了 traM 启动子的活性，而 6 个则没有影响。激活两个启动子所需的氨基酸残基可能接触RNA聚合酶a亚基的C端结构域，而仅激活traI启动子所需的残基可能接触另一个RNA聚合酶组分。
Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promoter, where TraR binds to a site centred 45 nucleotides upstream of the transcription start site, and of the traM promoter, where TraR binds a site centred 66 nucleotides upstream. Alteration of 18 residues blocked activity at the traI promoter. Of these, alteration at three positions impaired TraR abundance or DNA binding, leaving 15 residues that are specifically needed for positive control. Of these 15 residues, nine also blocked or reduced activity at the traM promoter, while six had no effect. Amino acid residues required for activation of both promoters probably contact the C-terminal domain of the RNA polymerase a subunit, while residues required only for traI promoter activation may contact another RNA polymerase component.