Ribosome Structure and Function

项目摘要 这个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tRNAs的偶联易位。它包括重要的 翻译阅读框架如何保留(或移动)以及翻译阅读框架如何 核糖体解旋酶解开结构的mRNA。我们的实验室独一无二地使用了 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。我们还在扩展我们的方法，以包括单分子光学 镊子方法，与布斯塔曼特实验室(加州大学伯克利分校)和Single- 分子FRET，与Ermolenko实验室(大学)合作罗切斯特)，以及 低温电子显微镜，与CHU实验室合作(斯坦福/SLAC)。 在以前的研究中，我们已经确定了陷阱易位的结构 中间体，这提供了令人意想不到的洞察力，了解了mRNA和 通过核糖体的tRNA与大小范围的构象变化相耦合 核糖体本身的结构。然后我们创建了FRET对，使我们能够相互关联 亚基间旋转、L1茎的移动和30s亚单位头域的旋转 MRNA和tRNA的运动。我们计划扩大搜索范围，以发现新的中间体 各州。在用尽了以前捕获易位中间体的策略后，我们将 使用一种新的方法，在所有五个结构中利用一组显性-致命突变 我们预计延伸因子EF-G的结构域将在不同的步骤中阻止易位。 开发一种新的荧光标记方法，使其能够进行位点特异性标记 将FRET对直接与核糖体RNA结合将克服单分子的技术障碍 核糖体动力学的研究，包括利用分子同时测量的研究 与布斯塔曼特小组合作，推动和担心核糖体的变化。最后， 我们设计了模型结构的mRNAs，这将为研究 信使核糖核酸解旋酶的作用机制及易位结构的确定 复合体在遇到和解开mRNA螺旋的过程中停滞不前。

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.