Ahmed M, Borsch C M, Taylor S S, Vázquez-Laslop N, Neyfakh A A
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago 60612.
J Biol Chem. 1994 Nov 11;269(45):28506-13.
Multidrug transporters are membrane proteins which, by an unknown mechanism, recognize diverse toxic compounds and efflux them from cells. We found that two substrates of the Bacillus subtilis multidrug transporter Bmr, rhodamine 6G and tetraphenylphosphonium (TPP), enhance Bmr expression at the level of transcription. Gene knock-out experiments demonstrated that an open reading frame located immediately downstream of the bmr gene is required for this enhancement. The protein product of this open reading frame, BmrR, shows distinct sequence homology to several known bacterial transcription activator proteins, such as MerR and TipAL. Gel-mobility shift and DNase protection assays indicated that BmrR binds specifically, as a dimer, to the bmr gene promoter. Furthermore, the affinity of this binding was enhanced by rhodamine and TPP, thus suggesting that these structurally dissimilar molecules interact directly with BmrR. Indeed, we found that BmrR bound rhodamine 6G stoichiometrically, one rhodamine molecule/BmrR dimer, and that TPP competed with rhodamine for this binding. Our results indicate that the enhancement of Bmr expression by some of its substrates is due to the ability of the regulatory protein, BmrR, to bind structurally dissimilar compounds resulting in enhanced transcription of the transporter gene.
多药转运蛋白是一类膜蛋白，其通过未知机制识别多种有毒化合物并将它们排出细胞。我们发现，枯草芽孢杆菌多药转运蛋白Bmr的两种底物，若丹明6G和四苯基鏻（TPP），在转录水平增强Bmr的表达。基因敲除实验表明，bmr基因下游紧邻的一个开放阅读框对于这种增强是必需的。这个开放阅读框的蛋白质产物BmrR，与几种已知的细菌转录激活蛋白，如MerR和TipAL，显示出明显的序列同源性。凝胶迁移率变动分析和DNA酶保护分析表明，BmrR作为二聚体特异性地结合到bmr基因启动子上。此外，若丹明和TPP增强了这种结合的亲和力，因此表明这些结构不同的分子直接与BmrR相互作用。事实上，我们发现BmrR化学计量地结合若丹明6G，即一个若丹明分子/BmrR二聚体，并且TPP与若丹明竞争这种结合。我们的结果表明，Bmr的一些底物对其表达的增强是由于调节蛋白BmrR结合结构不同化合物的能力，从而导致转运蛋白基因转录增强。
