DNA binding and ToxR responsiveness by the wing domain of TcpP, an activator of virulence gene expression in Vibrio cholerae
DNA binding and ToxR responsiveness by the wing domain of TcpP, an activator of virulence gene expression in Vibrio cholerae
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DOI：
10.1016/s1097-2765(03)00222-3
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发表时间：
2003-07-01
期刊：
影响因子：
16
通讯作者：
DiRita, VJ
中科院分区：
文献类型：
--
作者：
Krukonis, ES;DiRita, VJ
霍乱弧菌的毒力需要两种膜定位激活剂TcpP和ToxR激活toxT。我们分离了12个tcpP激活突变体，它们分为两类：不管是否存在ToxR， I类突变体都没有活性，而II类突变体在与ToxR共表达时表现出接近野生型的活性。大多数I类突变体在翼域有病变，通过与有翼的螺旋-螺旋-螺旋激活剂家族的同源性预测。在交联试验中，I类突变体与启动子DNA结合较差，并且在很大程度上不能与ToxR相互作用，而II类突变体与ToxR保持物理相互作用。一个突变体构建的体外结合DNA很差，但通过激活toxT对ToxR有反应，并保持了ToxR的相互作用。我们认为，对TcpP的功能来说，ToxR相互作用（而非DNA结合）是必不可少的，TcpP的翼结构域能够与ToxR接触，从而产生TcpP- rna聚合酶结合。
Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR. We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR. Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators. Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR. One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction. We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association.