Kamionka Annette, Bogdanska-Urbaniak Joanna, Scholz Oliver, Hillen Wolfgang
Lehstuhl für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
Nucleic Acids Res. 2004 Feb 4;32(2):842-7. doi: 10.1093/nar/gkh200. Print 2004.
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 approximately 10(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.
我们首次报道了反向四环素阻遏物（revTetR）的体外特性。在没有诱导剂脱水四环素（atc）的情况下，二聚体野生型阻遏物（TetR）与四环素操纵子tetO结合，从而实现紧密抑制。我们分离出了revTetR G96E L205S突变体，与TetR相反，它仅在atc存在时才与tetO结合。这种反向作用突变体被过量表达并纯化。通过电泳迁移率变动分析（EMSA）分析效应物和DNA结合特性，并通过荧光滴定和表面等离子体共振进行定量。revTetR与atcMg结合的缔合常数K(A)约为10(8) M(-1)，比TetR低四个数量级。TetR对tetO的亲和力为5.6 +/- 2 x 10(9) M(-1)，在atc存在下revTetR对tetO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和游离的revTetR，对DNA的亲和力均为4 +/- 1 x 10(5) M(-1)的低亲和力。因此，atc不是revTetR的二聚化剂。我们讨论了TetR和revTetR之间潜在的结构差异，这些差异可能是这种活性逆转的基础。
