TrIR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrIR:TraR dimers
TrIR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrIR:TraR dimers
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DOI：
10.1046/j.1365-2958.2001.02385.x
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发表时间：
2001-04-01
影响因子：
3.6
通讯作者：
Winans, SC
中科院分区：
文献类型：
--
作者：
Chai, Y;Zhu, J;Winans, SC
根癌农杆菌的章鱼碱型Ti质粒需要群体感应蛋白TraR和Tral以及可扩散的信息素3-氧代辛酰基高丝氨酸内酯（AAl）来调节接合转移所需的基因。TraR活性被称为TrlR的蛋白质抑制，TrlR与TraR的氨基酸1-181非常相似，但由于密码子182处的阅读框的移位而被截短。这种移码不影响氨基末端结构域的合成，氨基末端结构域被认为结合自诱导物并介导蛋白质二聚化，但消除了羧基末端DNA结合结构域的翻译。在这项研究中，我们发现TrlR，像TraR一样，在大肠杆菌中过表达时需要AAl来溶解，TrlR结合每个蛋白单体的一个分子的AAl，支持TraR的氨基末端结构域包含AAl结合位点的预测。纯化的TrlR阻断TraR的特异性DNA结合和tra启动子的转录，支持先前用全细胞进行的研究。将TrlR与TraR融合蛋白在大肠杆菌中共表达。在大肠杆菌中，这些蛋白质容易形成异二聚体复合物，其在DNA结合活性中无活性。这些数据支持以下假设：（i）TraR的氨基末端的一半结合AAl并介导蛋白质二聚化;（ii）TraR二聚体中的两个DNA结合结构域是稳定DNA结合所需的;和（iii）TrIR通过直接蛋白质-蛋白质相互作用阻断TraR。
Octopine-type Ti plasmids of Agrobacterium tumefaciens require the quorum-sensing proteins TraR and Tral and the diffusible pheromone 3-oxooctanoyl homoserine lactone (AAl) to regulate genes required for conjugal transfer. TraR activity is inhibited by a protein called TrlR, which closely resembles amino acids 1-181 of TraR but is truncated as a result of a shift in the reading frame at codon 182. This frameshift does not affect synthesis of the amino-terminal domain, which is thought to bind autoinducer and mediate protein dimerization, but abolishes translation of the carboxyl-terminal, DNA-binding domain. in this study, we show that TrlR, like TraR, requires AAl for solubility when overexpressed in Escherichia coli, TrlR bound one molecule of AAl per protein monomer, supporting the prediction that the amino-terminal domain of TraR contains the AAl binding site. Purified TrlR blocked TraR for both specific DNA binding and transcription of a tra promoter, supporting previous studies performed with whole cells. When TrlR end a TraR fusion protein were coexpressed in E. coli, these proteins readily formed heterodimeric complexes that were inactive in DNA-binding activity. These data support the hypotheses that (i) the amino-terminal half of TraR binds AAl and mediates protein dimerization; (ii) both DNA-binding domains in a TraR dimer are required for stable DNA binding; and (iii) TrlR blocks TraR by direct protein-protein interactions.