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首页> 外文期刊>Journal of bacteriology >Mutations in toxR and toxSThat Separate Transcriptional Activation from DNA Binding at the Cholera Toxin Gene Promoter
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Mutations in toxR and toxSThat Separate Transcriptional Activation from DNA Binding at the Cholera Toxin Gene Promoter

机译：toxR和toxS中的突变将霍乱毒素基因启动子上的DNA结合与转录激活分开

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ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in Vibrio cholerae. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12 toxR andtoxS positive control mutants producing variant ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in toxR specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven toxR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion oftoxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.
机译：ToxR和ToxS是整合膜蛋白，可激活霍乱弧菌中毒力基因的转录。 ToxR可以分为三个不同的域：N末端胞质DNA结合域，中央跨膜域和C末端周质域。据认为，ToxS通过周质相互作用增强ToxR介导的转录激活。通过P22攻击噬菌体选择DNA结合，结合对霍乱毒素基因转录的筛选，可以产生12个 toxR 和 toxS 阳性对照突变体，它们从产生变体ToxR蛋白分离到与霍乱毒素启动子结合但不能激活转录的toxRS 操纵子。 toxR 中的一个突变指定了预测的螺旋-环-螺旋DNA结合域中的E82K变化，并破坏了ToxR介导的激活。七个 toxR 突变包括移码和终止密码子引入周质结构域，这些突变中的六个似乎产生蛋白水解加工的较短形式的ToxR，这表明即使短的周质缺失也可以改变ToxR在周质中的折叠。 。删除 toxS 不会改变ToxR的稳态水平，并且发现在没有ToxS的情况下ToxR能够与DNA结合，即使它没有激活转录。但是，ToxS L33S变体使ToxR易于蛋白水解，这表明ToxS的天然功能是与ToxR形成复合物。因此，映射到ToxR胞质DNA结合结构域，周质结构域或ToxS的某些改变将DNA结合活性与激活剂功能分开。这些数据支持通过ToxS增强ToxR周质结构域的正确组装或稳定性的模型。 ToxS的这种类似伴侣的活性可能是转录激活复合物而不是ToxR-DNA复合物形成所必需的。

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