Ribosome Structure and Function

项目摘要 该项目的重点是了解潜在的分子机制， 蛋白质合成过程中mRNA和tRNA的偶联易位。它包括重要的 翻译阅读框架如何被保存（或移位）以及 核糖体解旋酶解旋结构化的mRNA。我们的实验室独特地使用了 生物化学，结构生物学，遗传学，FRET和计算方法来解决这些问题 具有挑战性的问题。我们还将我们的方法扩展到包括单分子光学 镊子的方法，与布斯塔曼特实验室（加州大学伯克利分校）和单- 分子FRET，与Ermolenko实验室（罗切斯特大学）合作，以及 低温电子显微镜，与Chiu实验室（斯坦福大学/SLAC）合作。 在以前的研究中，我们已经确定了陷阱易位的结构， 中间体，这提供了意想不到的见解如何运动的mRNA和 tRNA通过核糖体偶联到大规模和小规模的构象变化， 核糖体本身的结构。然后我们创造了FRET对， 亚基间旋转，L1柄的移动和30 S亚基头部结构域的旋转， mRNA和tRNA的运动。我们计划扩大这一搜索，以发现新的中间体 states.在用尽了以前的策略捕获易位中间体，我们将 使用一种新的方法，该方法利用了所有五种结构中的一组显性致死突变， 延伸因子EF-G的结构域，我们预计将阻止在不同的步骤易位。 开发一种新的荧光标记方法，允许位点特异性标记 FRET对直接核糖体RNA将克服技术障碍，单分子 核糖体动力学的研究，包括同时测量分子 力和FRET变化的核糖体，在合作与布斯塔曼特集团。最后， 我们已经设计了模型结构的mRNA，这将为研究 mRNA解旋酶的机制和确定易位的结构 复合物在遇到和解旋mRNA螺旋的过程中停滞。

Project Summary This project focuses on understanding the molecular mechanisms underlying the coupled translocation of mRNA and tRNAs during protein synthesis. It includes the important related problems of how the translational reading frame is preserved (or shifted) and how the ribosomal helicase unwinds structured mRNAs. Our laboratory uniquely uses a combination of biochemistry, structural biology, genetics, FRET and computational methods to address these challenging problems. We are also extending our approaches to include single-molecule optical tweezer methods, in collaboration with the Bustamante laboratory (UC Berkeley) and single- molecule FRET, in collaboration with the Ermolenko laboratory (Univ. of Rochester), as well as cryo-electron microscopy, in collaboration with the Chiu laboratory (Stanford/SLAC). In previous studies, we have determined the structures of trapped translocation intermediates, which have provided unexpected insights into how the movements of mRNA and tRNA through the ribosome are coupled to large- and small-scale conformational changes in the structure of the ribosome itself. We then created FRET pairs that allowed us to correlate intersubunit rotation, movement of the L1 stalk and rotation of the 30S subunit head domain with movements of mRNA and tRNA. We plan to extend this search to discover new intermediate states. Having exhausted previous strategies for trapping translocation intermediates, we will use a new approach which exploits a set of dominant-lethal mutations in all five structural domains of elongation factor EF-G that we expect will block translocation at different steps. Development of a novel fluorescent labeling approach that will allow site-specific labeling of FRET pairs directly to ribosomal RNA will overcome technical barriers to single-molecule studies of ribosome dynamics, including studies using simultaneous measurement of molecular forces and FRET changes in the ribosome, in collaboration with the Bustamante group. Finally, we have designed model structured mRNAs that will provide the basis for studying the mechanism of the mRNA helicase and for determination of the structures of translocation complexes stalled in the act of encountering and unwinding an mRNA helix.