Structural Robustness of Ribosome Functional Centers

描述（由申请人提供）：核糖体是蛋白质合成的通用位点，包含所有生物序列中一些最高度保守的序列。然而，核糖体对突变是强健的，在其高度保守的功能中心受到碱基或氨基酸取代的挑战时能够发挥作用。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位点。虽然已经确定抗生素耐药性突变具有巨大的适应性成本，但这种负担的结构基础现在才在我们的技术能力范围内进行调查。在这篇论文中，我们描述了一种利用遗传学、化学探测和x射线晶体学对嗜热细菌的核糖体进行合成的方法，以解决核糖体活性位点的结构稳健性及其与生物适应性的关系。嗜热T.核糖体遗传学的发展使我们能够随意识别或构建耐抗生素突变体。我们现在也有技术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。随着新型化学探测技术的发展，研究RNA构象动力学，这些能力使我们处于一个独特的位置，以解决三个具体问题。我们建议的第一个目的是使用链霉素耐药突变作为范式来检查参与30S亚基全局构象变化的保守核糖体功能中心的突变稳健性。我们的第二个目标是利用结核放线菌素耐药性来研究突变对亚基间桥的结构和功能的影响，亚基间桥对整个70S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的有害抗生素抗性突变来进化恢复适应性的补偿性突变，并使用x射线晶体学检查其结构效应。这个目的的目的是检测到尚未认识的远程功能关系，整个核糖体。我们还将使用肽基转移酶活性位点来检查核糖体功能中心对突变的稳健性的限制。除了在前所未有的分辨率水平上对抗生素耐药性提供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理。

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.