AdoMet-dependent tRNA methyl transferases

 描述（由申请人提供）：外排引起的多药耐药性是治疗细菌感染中最紧迫的问题之一。抗生素从细胞中挤出， 达到足够高的细胞内浓度以发挥治疗效果。虽然解决这一问题的努力集中在一次靶向一个外排泵，但耐药突变可能会迅速发展。我们建议靶向TrmD以同时减少多个泵的外排，从而加速杀菌作用。TrmD是一种细菌特异性S-腺苷甲硫氨酸（S-腺苷甲硫氨酸）依赖性tRNA甲基转移酶，控制阅读框上蛋白质合成的准确性。TrmD的缺失导致+1移码（+1FS）错误的积累，这导致蛋白质合成的过早终止。我们最近发现在大肠杆菌中存在多个外排基因。在大肠杆菌和其它革兰氏（-）细菌中，在阅读框的AUG起始密码子附近含有TrmD依赖性密码子。因此，我们假设靶向TrmD可以抑制所有这些基因的蛋白表达。通过同时减少多个泵的药物外排，我们提出靶向TrmD为未满足的医疗需求提供了一种新的解决方案。 成功靶向TrmD需要了解其m1 G37-tRNA结构域及其产物m1 G37-tRNA维持的质量控制机制。使用大肠coliTrmD（EcTrmD）为模型，我们在以下三个方面提供了这些前提条件。在目标1中，我们将开发一个分子水平的理解TrmD的cDNAMet结构域。该结构域具有不寻常的能力，即使在低水平的C3 Met下也能维持甲基化活性。我们将测试 这种能力是由结构域的不寻常拓扑蛋白结折叠赋予的假设，该结构域在结合时“弯曲”甲基供体。使用动力学和细胞分析，我们将确定蛋白质结折叠如何使用cDNAMet结合来促进tRNA结合并促进甲基转移。我们推测，这种轻松的协调之间的cDNAMet结合和催化活性是在该域的独特生物学的根源。在目标2中，我们将从机制层面理解TrmD的m1 G37-tRNA产物如何提高核糖体上蛋白质合成的准确性。准确性将由m1 G37-tRNA减少光滑mRNA序列的+1FS错误和减少cmo 5 U34的解码错误的能力决定，cmo 5 U34是一种在天然tRNA中经常伴随m1 G37的摆动修饰。在目标3中，我们将在细胞水平上了解TrmD的失活如何减少外排蛋白的合成。我们假设这种外排蛋白的减少将增加传统抗生素的细胞内积累，导致更快的杀菌肌动蛋白。我们还将使用核糖体分析来确定TrmD活性的范围，以确定其蛋白质表达在TrmD缺乏时被抑制的基因。这将为抗生素靶向的新策略和抗生素发现的新范式提供基础。

 DESCRIPTION (provided by applicant): Multi-drug resistance due to efflux is one of the most pressing issues in treating bacterial infections. Antibiotics are extruded from the cell and cannot reach high enough intracellular concentrations to exert a therapeutic effect. While efforts to address this problem have focused on targeting one efflux pump at a time, resistance mutations can quickly develop. We propose to target TrmD to reduce efflux at multiple pumps simultaneously so as to accelerate bactericidal action. TrmD is a bacterial-specific S-adenosyl-methionine (AdoMet)-dependent tRNA methyl transferase that controls the accuracy of protein synthesis on the reading frame. Loss of TrmD leads to accumulation of +1 frameshift (+1FS) errors, which cause pre-mature termination of protein synthesis. We recently discovered that multiple efflux genes in E. coli and in other Gram (-) bacteria contain TrmD-dependent codons near the AUG start codon of the reading frame. We therefore hypothesize that targeting TrmD can inactivate protein expression of all of these genes. By reducing drug efflux of multiple pumps at once, we propose that targeting TrmD offers a novel solution to an unmet medical need. Successful targeting TrmD requires an understanding of its AdoMet domain and the quality control mechanism maintained by its product m1G37-tRNA. Using E. coli TrmD (EcTrmD) as a model, we provide these prerequisites in the following three aims. In Aim 1, we will develop a molecular-level understanding of the AdoMet domain of TrmD. This domain has the unusual ability to maintain the methylation activity even at low levels of AdoMet. We will test the hypothesis that this ability is conferred by the unusual topological protein knot-fold of the domain that "bends" the methyl donor upon binding. Using both kinetic and cellular assays, we will determine how the protein knot-fold uses AdoMet binding to facilitate tRNA binding and to promote methyl transfer. We hypothesize that this facile coordination between AdoMet binding and catalytic activity is at the root of the unique biology of the domain. In Aim 2, we will develo a mechanistic-level understanding of how the m1G37-tRNA product of TrmD improves the accuracy of protein synthesis on the ribosome. Accuracy will be determined by the ability of m1G37-tRNA to reduce +1FS errors at slippery mRNA sequences and to reduce decoding errors of cmo5U34, a wobble modification that frequently accompanies m1G37 in natural tRNAs. In Aim 3, we will develop a cellular-level understanding of how inactivation of TrmD reduces synthesis of efflux proteins. We hypothesize that this reduction of efflux proteins will increase intracellular accumulation of traditional antibiotics, leading to faster bactericidal actin. We will also define the scope of TrmD activity using ribosome profiling to identify genes whose protein expression is arrested in TrmD deficiency. This will provide the basis for new strategies of antibiotic targeting and new paradigms for antibiotic discovery.