Luo Z Q, Qin Y, Farrand S K
Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
J Biol Chem. 2000 Mar 17;275(11):7713-22. doi: 10.1074/jbc.275.11.7713.
Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoserine lactone Agrobacterium autoinducer (AAI). Unique to this system, the activity of TraR is negatively modulated by an antiactivator called TraM. Analyses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact and the region of TraR responsible for this interaction are not known. Induction of traM in a strain in which TraR was activating transcription of a reporter system led to rapid cessation of gene expression. As assessed by a genetic assay that measures AAI-dependent DNA binding, TraM inhibited TraR function before and after the transcription factor had bound to its DNA recognition site. Consistent with this observation, in gel retardation assays, purified TraM abolished the DNA binding activity of TraR in a concentration-dependent manner. Such inhibition occurred independent of the order of addition of the reactants. As assessed by far Western analyses TraM interacts with TraR by directly binding the activator. TraM in its native form interacted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein. TraM interacted with TraR on blots prepared with total lysates of cells grown in the presence and absence of AAI. Far Western analysis of N- and C-terminal deletion mutants localized a domain of TraR contributing to TraM binding to the C-terminal portion of the activator protein. Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as for transcriptional activation or/and DNA binding. We conclude that TraM inhibits TraR by binding to the activator at a domain within or close to the helix-turn-helix motif located at the C terminus of the protein.
根癌土壤杆菌Ti质粒的接合转移受群体感应调控，通过转录激活因子TraR和酰基高丝氨酸内酯根癌土壤杆菌自诱导物（AAI）实现。该系统的独特之处在于，TraR的活性受到一种称为TraM的抗激活因子的负调控。酵母双杂交研究分析表明，TraM直接与激活因子相互作用，但这些组分相互作用的条件以及TraR中负责这种相互作用的区域尚不清楚。在TraR激活报告系统转录的菌株中诱导traM表达，会导致基因表达迅速停止。通过一种测量AAI依赖性DNA结合的遗传检测方法评估，TraM在转录因子结合到其DNA识别位点之前和之后均抑制TraR功能。与这一观察结果一致，在凝胶阻滞分析中，纯化的TraM以浓度依赖的方式消除了TraR的DNA结合活性。这种抑制作用与反应物添加顺序无关。通过远缘Western分析评估，TraM通过直接结合激活因子与TraR相互作用。天然形式的TraM与天然TraR相互作用，也与热处理后的TraR相互作用，但仅当变性蛋白中含有SDS时才会如此。TraM与在有和没有AAI的情况下生长的细胞总裂解物制备的印迹上的TraR相互作用。对N端和C端缺失突变体的远缘Western分析将TraR中有助于TraM结合的一个结构域定位到激活蛋白的C端部分。通过羟胺处理和易错聚合酶链反应进行随机诱变，确定了TraR这一区域中几个对于与TraM相互作用以及转录激活或/和DNA结合很重要的残基。我们得出结论，TraM通过在位于蛋白质C末端的螺旋-转角-螺旋基序内或附近的一个结构域结合激活因子来抑制TraR。
