Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain.
Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain.
复制标题
trp 阻遏物的 NH2 末端臂的突变分析表明有一个多功能结构域。
DOI：
10.1046/j.1365-2958.1998.00721.x
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发表时间：
1998
影响因子：
3.6
通讯作者：
Hurlburt,BK
中科院分区：
文献类型：
--
作者：
Mackintosh,SG;McDermott,PF;Hurlburt,BK
大肠杆菌阻遏蛋白的 NH2 末端臂涉及三种功能：通过与非操纵基因 DNA 结合形成阻遏蛋白-操纵基因复合物；与 DNA 结合的阻遏寡聚体的稳定性；以及在没有 DNA 的情况下脱孔阻抑物的寡聚化。为了开始检查负责这些不同活性的臂的结构方面，我们产生了一组广泛的缺失和取代突变体，并使用报告基因融合测量了这些突变体的体内活性。删除臂的任何部分都会导致 thetrp 和 thetrpRoperon 的阻遏蛋白活性显着降低。位置 4、5 和 6 对错义变化最敏感。这些位置上的大多数取代导致阻遏蛋白的活性低于野生型阻遏蛋白的 5%。大部分错义突变体在含有色氨酸的培养基中比野生型阻遏蛋白更活跃，而在不含色氨酸的培养基中活性较低。这种表型可以用阻遏蛋白和脱孔阻遏蛋白寡聚化的改变来解释。此外，还发现了九个超级阻遏突变体，这些突变体是由聚集在臂两端的取代产生的。我们的结果支持这样的假设，即 tr 预阻抑子的 NH2 末端臂是一个多功能结构域，并揭示了可能参与各种功能的结构成分。
The NH2‐terminal arms of theEscherichia coli trprepressor have been implicated in three functions: formation of repressor–operator complexes via association with non‐operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutantsin vivousing reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both thetrpand thetrpRoperons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild‐typetrprepressor. A large percentage of the missense mutants were more active than the wild‐type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super‐repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH2‐terminal arm of thetrprepressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.