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，含有所有生物序列中最高度保守的一些。尽管如此，核糖体对突变是稳健的，当在其高度保守的功能中心被碱基或氨基酸取代时能够发挥作用。作为抗生素的主要靶点，这些功能中心是许多抗生素抗性突变的位点。虽然已经很好地确定了抗虫性突变会带来相当大的适应性成本，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。在这个建议中，我们描述了一种合成方法，使用遗传学，化学探测和X-射线晶体学的核糖体从嗜热菌Thermus thermophilus来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T.嗜热菌核糖体遗传学使我们能够随意鉴定或构建抗嗜热菌突变体。我们现在也有技术能力结晶野生型和突变的30 S核糖体亚基和70 S核糖体，并确定其三维结构的X射线衍射。再加上开发新的化学探测技术来研究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。我们的建议的第一个目的是使用链霉素抗性突变作为一个范例，用于检查参与全球构象变化的30 S亚基的保守的核糖体功能中心的突变鲁棒性。我们的第二个目标是使用结核放线菌素抗性来研究突变对整个70 S核糖体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。第三个目的是利用有害的抗突变的肽基转移酶活性位点的补偿突变，恢复健身，并检查其结构的影响，使用X射线晶体学。这一目标的目的是检测尚未识别的整个核糖体的远程功能关系。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鲁棒性的极限。除了以前所未有的分辨率水平提供对抗生素耐药性的更完整的机械理解外，这些努力还旨在建立核糖体结构组织和进化的基本原则。

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.