STRUCTURAL ROBUSTNESS OF THE RIBOSOME

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 核糖体是蛋白质合成的通用场所，包含所有生物序列中最高度保守的一些。然而，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，能够发挥功能。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药性突变会带来巨大的适应性成本，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。在这里，我们将使用X-射线晶体学的核糖体从嗜热菌嗜热栖热菌，以解决结构的鲁棒性的核糖体活性位点和它的关系，生物健身。在与布朗大学的Albert Dahl-berg和Steven Gregory的合作中，我们将研究链霉素抗性突变，以确定参与30 S亚基全局构象变化的保守核糖体功能中心的突变稳健性。与Frank Murphy合作，我们已经收集了携带链霉素抗性突变和链霉素依赖性突变G524 U的30 S亚基晶体的差异数据集。总的来说，我们有携带几种抗生素抗性突变的30 S亚基的晶体，我们正在制备大量额外的核糖体样品。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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 chal-lenged 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. Here, we will use 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. In collaboration with Albert Dahl-berg and Steven Gregory at Brown University, we will investigate streptomycin-resistance mutations to ex-amine the mutational robustness of a conserved ribosome functional center that participates in global con-formational changes of the 30S subunit. In collaboration with Frank Murphy, we have already collected dif-fraction data sets for 30S subunit crystals carrying streptomycin-resistance mutations and the streptomycin-dependence mutation G524U. Overall, we have crystals available for 30S subunits carrying several antibi-otic-resistance mutations and we are in the process of preparing a large number of additional ribosome sam-ples. In addition to providing a more complete mechanistic understanding of antibiotic resistance at an un-precedented level of resolution, these efforts are directed towards establishing fundamental principles of ri-bosome structural organization and evolution.