Molecular Characterization of GrlA, a Specific Positive Regulator of ler Expression in Enteropathogenic Escherichia coli
Molecular Characterization of GrlA, a Specific Positive Regulator of ler Expression in Enteropathogenic Escherichia coli
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DOI：
10.1128/jb.00307-10
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发表时间：
2010-09-01
影响因子：
3.2
通讯作者：
Puente, Jose L.
中科院分区：
文献类型：
--
作者：
Jimenez, Rafael;Cruz-Migoni, Sara B.;Puente, Jose L.
肠致病性大肠杆菌（EPEC）感染的特征是在感染的上皮细胞表面形成附着和消退（A/E）损伤。形成A/E病变所需的基因位于肠上皮细胞消失（LEE）的位点内。Ler是控制LEE基因表达的关键调控因子。ler基因的表达受LEE编码的GrlA正调控。在这里，我们分析的机制，GrlA积极调节LER的表达，并表明，在没有H-NS，GrlA不再是必要的LER激活，进一步证实GrlA作为一个H-NS拮抗剂的LER启动子的一部分。构建单氨基酸突变体以测试在GrlA的N-末端一半以及蛋白质的C-末端结构域中发现的推定螺旋-转角-螺旋（HTH）DNA结合基序的功能意义。HTH基序中的几个突变，但不是全部，完全消除了GrlA活性，以及与Ier调节区中位置-54下游的靶序列的特异性结合。这些突变体中的一些，虽然没有活性，但仍然能够与负调节因子GrlR相互作用，表明活性的丧失不是蛋白质错误折叠的结果。HTH结构域附近以及蛋白质末端的其他残基也被证明对于GrlA作为转录调节因子的活性是重要的，但对于其与GrlR的相互作用不是重要的。总之，GrlA由至少两个功能结构域组成，一个参与转录激活和DNA结合，另一个参与与GrlR的异源二聚化。
Enteropathogenic Escherichia coli (EPEC) infections are characterized by the formation of attaching and effacing (A/E) lesions on the surfaces of infected epithelial cells. The genes required for the formation of A/E lesions are located within the locus of enterocyte effacement (LEE). Ler is the key regulatory factor controlling the expression of LEE genes. Expression of the ler gene is positively regulated by GrlA, which is encoded by the LEE. Here, we analyze the mechanism by which GrlA positively regulates ler expression and show that in the absence of H-NS, GrlA is no longer essential for ler activation, further confirming that GrlA acts in part as an H-NS antagonist on the ler promoter. Single-amino-acid mutants were constructed to test the functional significance of the putative helix-turn-helix (HTH) DNA binding motif found in the N-terminal half of GrlA, as well as at the C-terminal domain of the protein. Several mutations within the HTH motif, but not all, completely abolished GrlA activity, as well as specific binding to its target sequence downstream from position -54 in the ler regulatory region. Some of these mutants, albeit inactive, were still able to interact with the negative regulator GrlR, indicating that loss of activity was not a consequence of protein misfolding. Additional residues in the vicinity of the HTH domain, as well as at the end of the protein, were also shown to be important for GrlA activity as a transcriptional regulator, but not for its interaction with GrlR. In summary, GrlA consists of at least two functional domains, one involved in transcriptional activation and DNA binding and the other in heterodimerization with GrlR.