Research Group of Microbiology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussel, Belgium ; Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Centro de Investigación Biomédicaen Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, E-46010 Valencia, Spain.
Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Centro de Investigación Biomédicaen Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, E-46010 Valencia, Spain.
FEBS Open Bio. 2015 Jan 28;5:76-84. doi: 10.1016/j.fob.2015.01.002. eCollection 2015.
RutR is a member of the large family of TetR transcriptional regulators in Escherichia coli. It was originally discovered as the regulator of the rutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of the carAB operon, encoding carbamoylphosphate synthase. Unlike most other TetR-like regulators, RutR exerts both positive and negative effects on promoter activity. Furthermore, RutR exhibits a very narrow ligand binding specificity, unlike the broad effector specificity that characterizes some of the well-studied multidrug resistance regulators of the family. Here we focus on ligand binding and ligand specificity of RutR. We construct single alanine substitution mutants of amino acid residues of the ligand-binding pocket, study their effect on in vitro DNA binding in absence and presence of potential ligands, and analyse their effect on positive regulation of the carP1 promoter and negative autoregulation in vivo. Although RutR structures have been determined previously, they were deposited in the Protein Data Bank without accompanying publications. All of them have uracil bound in the effector-binding site, representing the inactive form of the regulator. We determined the crystal structure of an unliganded mutant RutR protein and provide a structural basis for the use of uracil as sole effector molecule and the exclusion of the very similar thymine from the ligand-binding pocket.
RutR 是大肠杆菌中 TetR 转录调节因子大家族的成员。它最初是作为编码嘧啶利用新途径的 rutABCDEFG 操纵子的调节子被发现的，但它的最高亲和力靶标是编码氨甲酰磷酸合酶的 carAB 操纵子的控制区。与大多数其他 TetR 样调节剂不同，RutR 对启动子活性既具有正调节作用，又具有负调节作用。此外，RutR 表现出非常狭窄的配体结合特异性，与该家族中一些研究得很好的多药耐药调节剂的广泛效应物特异性不同。在这里，我们重点研究 RutR 的配体结合和配体特异性。我们构建了配体结合口袋氨基酸残基的单个丙氨酸取代突变体，研究了它们在不存在和存在潜在配体的情况下对体外 DNA 结合的影响，并分析了它们对 carP1 启动子的正调节和体内负自调节的影响。尽管先前已经确定了 RutR 结构，但它们在没有伴随出版物的情况下被存入蛋白质数据库。它们都在效应物结合位点结合了尿嘧啶，代表了调节剂的无活性形式。我们确定了一种未配体结合的突变 RutR 蛋白的晶体结构，并为将尿嘧啶作为唯一效应分子的使用以及将非常相似的胸腺嘧啶排除在配体结合口袋之外提供了结构基础。
