Atomic-resolution structure and function of the ribosome

描述(申请人提供)：核糖体在所有形式的生命中，在将基因型转化为表型的过程中起着关键作用。它是负责蛋白质合成的大型RNA和蛋白质工厂，将信使RNA中的遗传密码翻译成相应的蛋白质。最近对核糖体的生化和结构研究已经开始揭示翻译的分子基础。这两个核糖体亚基的图像已经通过X射线结晶学在原子分辨率上分别确定。然而，单个核糖体亚基缺乏蛋白质合成所必需的核糖体的许多功能。核糖体亚基必须作为一个完整的核糖体一起工作，才能发生翻译。 为了在分子水平上提供蛋白质合成周期的完整图景，有必要对完整的核糖体进行原子分辨率的“电影”，将氨基酸添加到不断增长的多肽链中。我们建议制作这部电影的第一帧，通过确定模式生物大肠杆菌中两个完整核糖体的原子分辨率结构。通过关注大肠杆菌核糖体，我们将能够直接将我们的结构结果与几十年来使用这个模型系统进行的关于翻译的生化和遗传学研究进行比较。 在这笔赠款的头四年里，我们在9-12A的分辨率下测定了整个大肠杆菌核糖体的X射线晶体结构，这揭示了翻译的结构基础。这些结构为我们的目标提供了第一步，即在蛋白质延伸周期中以原子分辨率制作核糖体的“快照”。我们现在已经获得了完整的大肠杆菌核糖体的晶体，它可以将X射线衍射到原子分辨率。这些晶体将使我们第一次能够以原子分辨率对核糖体进行成像，并以原子细节探索核糖体突变对抗生素耐药性的影响。我们的结果将显著影响我们对翻译的理解，并将在翻译发挥重要作用的许多生物学领域广泛使用。该建议的具体目标如下： 1.从大肠杆菌中分离并提纯两个完整的70年代核糖体的原子分辨结构。 2.确定两种大肠杆菌核糖体结构的功能状态。 3.探索导致抗生素耐药性的核糖体突变的结构和功能后果。

DESCRIPTION (provided by applicant): The ribosome plays a key role in the conversion of genotype into phenotype in all forms of life. It is the large RNA and protein factory responsible for protein synthesis, and translates the genetic code in messenger RNA into the corresponding protein. Recent biochemical and structural studies of the ribosome have started to reveal the molecular basis of translation. Images of the two ribosomal subunits have been determined individually at atomic resolution by x-ray crystallography. However, the individual ribosomal subunits lack many functions of the ribosome necessary for protein synthesis. The ribosomal subunits must work together as an intact ribosome, in order for translation to occur. In order to provide a complete picture of the protein synthesis cycle at the molecular level, an atomic resolution "movie" of the intact ribosome, adding an amino acid to a growing polypeptide chain, will be necessary. We propose to make the first "frames" of this movie, by determining the atomic-resolution structures of two intact ribosomes from the model organism, Escherichia coli. By focusing on the E. coli ribosome, we will be able to compare our structural results directly to decades of biochemical and genetic research on translation, that has been carried out with this model system. During the first four years of this grant, we determined x-ray crystal structures of the entire  coli ribosome at resolutions of 9-12 A, that shed some light on the structural basis of translation. These structures provide a first step in our goal of making "snapshots" of the ribosome at atomic resolution during the protein elongation cycle. We have now obtained crystals of the intact E. coli ribosome that diffract x-rays to atomic resolution. These crystals will allow us, for the first time, to image the ribosome at atomic resolution, and to explore, in atomic detail, the impact of ribosomal mutations that confer antibiotic resistance. Our results will significantly impact our understanding of translation, and will be widely useful in many biological fields where translation plays an important role. The specific aims of the proposal are as follows: 1. Solve and refine atomic-resolution structures of two intact 70S ribosomes from E. coli. 2. Determine the functional states of the two  coli ribosome structures. 3. Probe the structural and functional consequences of mutations in the ribosome that cause antibiotic resistance.