Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor
Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor
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DOI：
10.1093/nar/gkh200
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Hillen, W
中科院分区：
文献类型：
--
作者：
Kamionka, A;Bogdanska-Urbaniak, J;Hillen, W
我们首次报告了反向四环素阻遏物（revTetR）的体外表征。在缺乏诱导剂脱水四环素 (atc) 的情况下，二聚体野生型阻遏蛋白 (TetR) 与 tet 操纵子 tetO 结合，从而产生严格的阻遏作用。我们分离出了 revTetR G96E L205S 突变体，与 TetR 相反，它仅在 atc 存在的情况下结合 tetO。这种反向作用突变体被过量生产和纯化。通过 EMSA 分析效应子和 DNA 结合特性，并通过荧光滴定和表面等离子共振进行定量。 revTetR与[atcMg](+)结合的缔合常数K-A与10(8)M-1相似，比TetR低4个数量级。 TetR 对 tetO 的亲和力为 5.6 +/- 2 x 10(9) M-1，在 atc 存在下对 revTetR 的亲和力为 1 +/- 0.2 x 10(8) M-1。两种诱导形式，即 atc 结合的 TetR 和游离 revTetR，对 DNA 具有相同的低亲和力，均为 4 +/- 1 x 10(5) M-1。因此，atc 不充当 revTetR 的二聚剂。我们讨论了 TetR 和 revTetR 之间的结构差异可能是这种活性逆转的基础。
We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant K-A of revTetR for binding of [atcMg](+) is similar to10(8) M-1, four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 +/- 2 x 10(9) M-1 and that for revTetR in the presence of atc is 1 +/- 0.2 x 10(8) M-1. Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 +/- 1 x 10(5) M-1 for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.