Saenger W, Orth P, Kisker C, Hillen W, Hinrichs W
Freie Universität Berlin Institut für Kristallographie Takustrasse 6, 14195 Berlin (Germany).
Angew Chem Int Ed Engl. 2000 Jun 16;39(12):2042-2052. doi: 10.1002/1521-3773(20000616)39:12<2042::aid-anie2042>3.0.co;2-c.
The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex MgTc binds to (TetR)(2) to form the induced complex (TetR small middle dotMgTc)(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two MgTc to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dotMgTc)(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/MgTc system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.
抗生素的过度使用使细菌通过多种机制产生了耐药性。广谱抗生素四环素（Tc）最常见的抑菌作用是使细菌核糖体失活，从而中断蛋白质生物合成，导致细菌死亡。革兰氏阴性菌对Tc产生耐药性的最常见机制与膜内在蛋白TetA有关，TetA可在入侵的Tc攻击其靶标核糖体之前将其输出细菌细胞。TetA的表达受到同二聚体Tet阻遏蛋白（TetR）的严格调控，TetR与位于质粒或转座子上tetA基因上游的操纵子DNA（tetO；结合常数约为10¹¹ M⁻¹）的两个螺旋-转角-螺旋基序特异性结合。当Tc扩散到细胞中时，它会螯合Mg²⁺，形成的复合物[MgTc]⁺与（TetR）₂结合形成诱导复合物（TetR·[MgTc]⁺）₂。这个过程伴随着构象变化，这会大幅降低（TetR）₂对tetO的亲和力，从而使TetA得以表达，赋予细菌细胞对Tc的耐药性。晶体学研究表明了（TetR）₂·tetO复合物中序列特异性的蛋白质-核酸相互作用，以及两个[MgTc]⁺与（TetR）₂的结合如何促使构象变化，这种变化通过每条链上八个水分子的协同结合而稳定下来，使得形成的（TetR·[MgTc]⁺）₂不再能够识别并结合tetO。由于TetR/[MgTc]⁺系统的转换机制非常紧密，它在真核基因表达调控中已被证明非常有用，也可能适用于基因治疗。
