The role of the variable region in Tet repressor for inducibility by tetracycline
The role of the variable region in Tet repressor for inducibility by tetracycline
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DOI：
10.1074/jbc.272.11.6936
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发表时间：
1997-03-14
影响因子：
4.8
通讯作者：
Hillen, W
中科院分区：
文献类型：
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作者：
Berens, C;Schnappinger, D;Hillen, W
在Tn10Tet阻遏物(TetR)的α8和α9螺旋之间引入了一组丙氨酸的缺失和取代。这个区域在TetR(D).([mg-TC](+))(2)复合体的晶体结构中呈现为一个非结构化的环，是来自七个不同抗性决定因素的Tet阻遏物排列中唯一长度可变的内部片段。对10个突变体的体内分析表明，这个环对四环素(TC)的诱导性很重要，而DNA结合没有或仅受到轻微影响，所有缺失都具有诱导缺陷的TetR(S)表型，但相应的替换不或仅轻微影响诱导性。纯化的突变体TetR蛋白在体外对TC的亲和力降低，这与它们缺乏诱导性有关。[mg-TC](+)与TetR突变体的结合率提高。由于没有一个突变残基在晶体结构中与TC直接接触，我们认为环的长度对于TetR的闭合的TC结合构象和开放的操作符结合构象之间的结构转换是重要的。我们认为，环中的缺失使两种形式之间的平衡向开放的、操作符结合的构象移动。
A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha 8 and alpha 9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc](+))(2) complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected, All deletions have an induction-deficient TetR(S) phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc](+) to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.