Structural Robustness of Ribosome Functional Centers

描述(申请人提供)：核糖体是蛋白质合成的通用部位，包含所有生物序列中最保守的一些。然而，核糖体对突变是强大的，能够在其高度保守的功能中心受到碱基或氨基酸替换的挑战时发挥功能。作为抗生素的主要靶点，这些功能中心是许多抗生素耐药突变的地点。虽然抗生素耐药性突变会带来巨大的适应成本，但这种负担的结构基础现在才在我们的技术能力调查范围内。在这项建议中，我们描述了一种利用遗传学、化学探测和X射线结晶学对嗜热细菌Thermus thermophilus的核糖体进行合成的方法，以解决核糖体活性部位的结构稳定性及其与生物适合性的关系。我们对嗜热梭菌核糖体遗传学的发展使我们能够随意鉴定或构建耐药突变株。我们现在也有能力结晶野生型和突变型30S核糖体亚基和70S核糖体，并通过X射线衍射确定它们的三维结构。再加上研究RNA构象动力学的新的化学探测技术的发展，这些能力使我们在解决三个具体问题方面处于独特的地位。我们建议的第一个目标是使用链霉素耐药突变作为检测参与30S亚基全球构象变化的保守核糖体功能中心突变稳健性的范例。我们的第二个目标是利用对结核放线菌素的抗药性来检查突变对亚基间桥的结构和功能的影响，亚基间桥对整个70年代核糖体的大规模旋转运动至关重要。第三个目标是利用肽基转移酶活性位点上的有害抗生素耐药性突变来进化出恢复健康的补偿突变，并使用X射线结晶学来检查它们的结构效应。这一目标的目的是检测整个核糖体中尚未被识别的长程功能关系。我们还将使用肽基转移酶活性位点来检测核糖体功能中心对突变的稳健性极限。除了在前所未有的分辨率水平上提供对抗生素耐药性的更完整的机制理解外，这些努力还旨在建立核糖体结构组织和进化的基本原则。 公共卫生相关性：该项目的目标是研究抗生素耐药突变对核糖体结构和功能的影响，以更好地了解耐药的分子机制。这些研究的结果将为合理开发新的核糖体靶向抗生素化合物提供有价值的信息，以对抗对现有药物具有耐药性的病原体。

DESCRIPTION (provided by applicant): The ribosome is the universal site of protein synthesis, containing some of the most highly conserved of all biological sequences. Nevertheless, the ribosome is robust to mutation, capable of functioning when challenged with base or amino acid substitutions in its highly conserved functional centers. As major targets of antibiotics, these functional centers are the sites of numerous antibiotic-resistance mutations. While it has been well established that antibiotic-Resistance mutations carry a substantial fitness cost, the structural basis for this burden is only now within the scope of our technical ability to investigate. In this proposal, we describe a synthetic approach using genetics, chemical probing and X-ray crystallography of ribosomes from the thermophilic bacterium Thermus thermophilus to address the structural robustness of ribosome active sites and its relationship to biological fitness. Our development of T. thermophilus ribosome genetics has enabled us to identify or construct antibiotic-resistant mutants at will. We also now have the technical ability to crystallize wild-type and mutant 30S ribosomal subunits and 70S ribosomes and to determine their three-dimensional structures by X-ray diffraction. Together with the development of novel chemical probing techniques to investigate RNA conformational dynamics, these abilities have placed us in a unique position to address three specific issues. The first aim of our proposal is to use streptomycin-resistance mutations as a paradigm for examining the mutational robustness of a conserved ribosome functional center that participates in global conformational changes of the 30S subunit. Our second aim is to use tuberactinomycin-resistance to examine the effects of mutations on the structure and function of an intersubunit bridge that is critical for large-scale rotational motions of the entire 70S ribosome. The third aim is to use deleterious antibiotic-resistance mutations in the peptidyltransferase active site to evolve compensatory mutations that restore fitness, and to examine their structural effects using X-ray crystallography. The goal of this aim is to detect as yet unrecognized long-range functional relationships throughout the ribosome. We will also use the peptidyltransferase active site to examine the limits of robustness of ribosome functional centers to mutation. In addition to providing a more complete mechanistic understanding of antibiotic resistance at an unprecedented level of resolution, these efforts are directed towards establishing fundamental principles of ribosome structural organization and evolution. PUBLIC HEALTH RELEVANCE: The goal of this project is to study the impact of antibiotic-resistance mutations upon ribosome structure and function in order to gain a better understanding of the molecular mechanism of resistance. Results from these studies will provide valuable information for the rational development of new ribosome-targeting antibiotic compounds to combat pathogens that are resistant to currently available drugs.