Structural Robustness of Ribosome Functional Centers

核糖体功能中心的结构稳健性

• 批准号: 7944383
• 负责人: Steven Gregory
• 金额: $ 30.78万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7944383
• 关键词: Active Sites; Address; Amino Acid Sequence; Amino Acid Substitution; Antibiotic Resistance; Antibiotics; Binding; Biological; Biological Assay; Biological Process; Biology; Chemicals; Complement; Data; Development; Equilibrium; Evolution; Genes; Genetic; Goals; Hybrids; In Vitro; Mass Spectrum Analysis; Measures; Methods; Molecular; Molecular Conformation; Motion; Mutation; Peptidyltransferase; Pharmaceutical Preparations; Positioning Attribute; Protein Biosynthesis; RNA; Resistance; Resolution; Ribosomal RNA; Ribosomes; Roentgen Rays; Signal Transduction; Site; Streptomycin; Structure; Techniques; Thermus thermophilus; Transfer RNA; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; chemical genetics; combat; cost; design; fitness; mutant; novel; pathogen; public health relevance; research study; resistance mechanism; resistance mutation; thermophilic bacteria; three dimensional structure; transmission process; tuberactinomycin

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.

描述（由申请人提供）：核糖体是蛋白质合成的通用部位，其中包含所有生物序列中一些最高度保守的。然而，核糖体对突变具有鲁棒性，在其高度保守的功能中心中用碱或氨基酸取代挑战时能够发挥功能。作为抗生素的主要靶标，这些功能中心是众多抗生素耐药突变的部位。虽然已经很好地确定抗生素抗性突变具有巨大的适应性成本，但这种负担的结构性基础仅在我们调查技术能力的范围内。在此提案中，我们描述了一种使用遗传学，化学探测和X射线晶体学对嗜热细菌嗜热细菌的核糖体的合成方法，以解决核糖体活性位点的结构鲁棒性及其与生物适应性的关系。 我们对嗜热链球菌核糖体遗传学的发展使我们能够随意识别或构建抗生素耐药突变体。现在，我们还具有将野生型和突变体30S核糖体亚基和70S核糖体结晶的技术能力，并通过X射线衍射来确定其三维结构。随着新型化学探测技术的发展以研究RNA构象动力学，这些能力使我们处于解决三个特定问题的独特位置。我们建议的第一个目的是使用链霉素抗性突变作为检查参与30S亚基的全局构象变化的保守核糖体功能中心的突变鲁棒性的范例。我们的第二个目的是使用结核霉素的抗性来检查突变对整个70年代核糖体的大规模旋转运动至关重要的亚基间桥的结构和功能的影响。第三个目的是在肽基转移酶的活性位点使用有害的抗生素耐药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。该目标的目的是检测整个核糖体的尚未认可的远程功能关系。我们还将使用肽基转移酶的活性位点来检查核糖体功能中心对突变的鲁棒性限制。除了在前所未有的分辨率水平上提供对抗生素抗性的更完整的机械理解外，这些努力还针对建立核糖体结构组织和进化的基本原理。 公共卫生相关性：该项目的目的是研究抗生素耐药突变对核糖体结构和功能的影响，以便更好地了解抗性分子机制。这些研究的结果将为新的核糖体抗生素化合物的合理发展提供有价值的信息，以打击对当前可用药物具有抗性的病原体。