Context-specific action of antibiotics targeting the catalytic center of the bacterial ribosome

通过干扰蛋白质合成来抑制细胞生长的抗生素一直是最多的 临床上成功的抗菌药。尽管这些抑制剂很重要，但在以下方面存在显著差距 我们对他们行动的最基本原则的理解。许多蛋白质合成抑制剂， 从经典的氯霉素(CHL)到新的利奈唑胺(LZD)，结合在催化肽转移酶上 核糖体的中心(PTC)，在那里它们与氨基酰-tRNA的放置发生冲突。因为 其结合部位的位置，通常认为它们通过干扰形成 每个多肽键，要么位于基因的起始密码子，要么位于基因的任何内部密码子。然而，我们的 初步数据显示，这种观点基本上是不正确的。而不是不分青红皂白地抑制肽键 形成、CHL和LZD仅在特定的信使核糖核酸位点停止翻译延伸。新生生物的本质 多肽链似乎在确定翻译停滞的位置方面发挥了主要作用，但一般规则 关于失速位置的定义和这种效应背后的分子机制尚不清楚。 因此，本项目的主要目标是详细了解上下文中的具体操作 PTC靶向抗生素。这项研究将主要集中在LZD和CHL。LZD是第一个也是范围最广的 医用恶唑烷酮。Chl是已知最古老的核糖体抗生素之一。尽管它减少了 医学上的重要性，将CHL纳入研究是至关重要的，不仅因为大量的信息 可用于该抑制剂，但也用于将其上下文特定的作用与LZD的作用进行对比，并将 与药物的个别结构性质有关的作用。 在具体目标1中，将使用全细胞核糖体图谱和定量生化测试来 确定定义首选抑制部位的序列上下文的详细要求 由LZD或CHL翻译。在具体目标2中，一系列生化、结构和遗传方法将 被用来理解解释PTC的上下文特定作用的分子机制- 靶向抑制剂。创新技术的使用，如单分子FRET或工程技术 被拴住的核糖体，预计将提供主要对最基本的方面的新见解 核糖体催化中心抑制剂的作用。具体目标3将涉及一个概念上的重要问题 以及与医学相关的问题，药物作用的背景特异性是否会导致蛋白质特异性抑制 PTC靶向抗生素的翻译。 预期的调查结果应会大大扩大对以下行动的一般模式的理解 临床上重要的抗菌药，作用于核糖体的催化中心，并可能打开新的场所 用于合理开发具有优异抗菌性能的蛋白质合成抑制剂。

Antibiotics that inhibit cell growth by interfering with protein synthesis have been among the most clinically successful antibacterials. In spite of the importance of these inhibitors, there are significant gaps in our understanding of the most fundamental principles of their action. Many of the protein synthesis inhibitors, from the classic chloramphenicol (CHL) to the newer linezolid (LZD), bind at the catalytic peptidyl transferase center (PTC) of the ribosome, where they clash with the placement of aminoacyl-tRNA. Because of the location of their binding site, it is commonly assumed that they inhibit translation by interfering with formation of every peptide bond, either at the start codon or at any of the internal codons of a gene. However, our preliminary data show that this view is principally incorrect. Instead of indiscriminately inhibiting peptide bond formation, CHL and LZD stall elongation of translation only at specific mRNA sites. The nature of the nascent peptide chain appears to play the major role in specifying the sites of translation arrest, but the general rules that define the sites of stalling and the molecular mechanisms that underlie this effect remain unknown. Therefore, the main goal of this project is to gain a detailed understanding of the context specific action of PTC-targeting antibiotics. The study will primarily focus on LZD and CHL. LZD is the first and most broadly medically used oxazolidinone. CHL is one of the oldest known ribosomal antibiotics. In spite of its reduced medical importance, inclusion of CHL in the study is crucial, not only because of the vast amount of information available for this inhibitor, but also to contrast its context specific action with that of LZD and correlate the effects with individual structural properties of the drugs. In Specific Aim 1, whole-cell ribosome profiling and quantitative biochemical testing will be used to identify the detailed requirements for the sequence context that defines the preferred sites of inhibition of translation by LZD or CHL. In Specific Aim 2, an array of biochemical, structural and genetic approaches will be employed to understand the molecular mechanisms that account for the context-specific action of the PTC- targeting inhibitors. The use of innovative techniques, such as single molecule FRET or an engineered tethered ribosome, are expected to provide principally new insights into the most fundamental aspects of action of the inhibitors of the ribosomal catalytic center. Specific Aim 3 will address a conceptually important and medically-relevant question, whether context specificity of drug action results into protein-specific inhibition of translation by the PTC-targeting antibiotics. The anticipated findings should significantly expand the understanding of the general mode of action of clinically-important antibacterials that act upon the catalytic center of the ribosome and may open new venues for rational development of protein synthesis inhibitors with superior antibiotic properties.