The structural dynamics of ribosomal frameshifting and ribosome rescue

项目总结 核糖体和翻译的其余部分将信使RNA(MRNAs)翻译成蛋白质 机械(TM)是对生命至关重要的基因表达的基本步骤。因为细菌TM是一种 已被证明是开发新抗生素的目标，因为许多人类疾病都是 与翻译的失调有关，细菌和细菌的翻译和翻译控制的机制 真核生物仍在接受严密的调查。在过去的二十年里，结构研究揭示了 TM在蛋白质合成过程中经历了大规模的结构重排。不幸的是，大小， TM的复杂性和构象灵活性极大地阻碍了对这些动力学的研究， 极大地限制了我们对这些动力如何促进翻译机制的理解 翻译控制。尽管如此，使用单分子荧光和结构的组合 技术，我们和其他人已经能够描述翻译的核心步骤的动态 细菌TM.尽管取得了这些成就，但在我们的理解中，关于是否和 作为生物医学上重要的翻译控制的一部分，这些核心步骤的动力学是如何被调节的 战略。为了填补这些空白，在本应用程序的第一个目标中，我们建议使用单一- 分子荧光、结构和生物化学方法来研究细胞的动力学 细菌TM被调节以驱动核糖体移码。移帧是一种平移控制 TM向后或向前滑动mRNA上的一个或多个核苷酸以校正 一种插入或缺失的‘移码’突变，否则将导致异常或 截短的蛋白质或驱动一个单一的信使核糖核酸合成一个以上的蛋白质产物。这些 实验有望揭示框架转换背后仍然难以捉摸的机制(S)。在第二个目标中，我们 将使用类似的方法来研究核糖体救援因子如何调节 细菌TM作为识别和拯救核糖体机制的一部分 在翻译上受到损害。这些研究将提供基于结构的机械模型 细菌核糖体拯救系统，可用于开发新的抗生素。在第三个目标中， 我们将把单分子荧光和结构技术的组合扩展到酵母翻译 系统，使我们能够研究真核生物特有的核心步骤的翻译，移码， 和核糖体拯救。这些研究的结果将揭示驱动和调节 真核生物中的翻译，为研究人类翻译控制的作用提供了一个框架 健康和疾病。

PROJECT SUMMARY Translation of messenger RNAs (mRNAs) into proteins by the ribosome and the rest of the translation machinery (TM) is a fundamental step in gene expression that is central to life. Because the bacterial TM is a proven target for the development of new antibiotics and because many human diseases have been causally linked to dysregulation of translation, the mechanisms of translation and translational control in bacteria and eukaryotes remain under intense investigation. Over the past two decades, structural studies have revealed the large-scale structural rearrangements the TM undergoes during protein synthesis. Unfortunately, the size, complexity, and conformational flexibility of the TM have greatly impeded studies of these dynamics, significantly limiting our understanding of how these dynamics contribute to the mechanisms of translation and translational control. Nonetheless, using a combination of single-moleucle fluorescence and structural techniques, we and others have been able to characterize the dynamics of the core steps of translation by the bacterial TM. Despite these accomplishments, critical gaps in our understanding remain regarding whether and how the dynamics of these core steps are modulated as part of biomedically important translational control strategies. To fill these gaps, in the first aim of this application, we propose to use a combination of single- molecule fluorescence, structural, and biochemical approaches to investigate how the dynamics of the bacterial TM are modulated in order to drive ribosomal frameshifting. Frameshifting is a translational control strategy in which the TM slips backward or forward by one or more nucleotides on the mRNA to either correct an insertion or deletion ‘frameshift’ mutation that would otherwise result in production of an aberrant or truncated protein or to drive the synthesis of more than one protein product from a single mRNA. These experiments promise to reveal the still-elusive mechanism(s) that underlie frameshifting. In the second aim, we will use analogous approaches to investigate how ribosome rescue factors modulate the dynamics of the bacterial TM as part of the mechanisms through which they recognize and rescue ribosomes that have become translationally compromised. These studies will provide structure-based mechanistic models of bacterial ribosome rescue systems that can be exploited in the development of new antibiotics. In the third aim, we will extend our combination of single-molecule fluorescence and structural techniques to a yeast translation system, enabling us to investigate eukaryotic-specific aspects of the core steps of translation, frameshifting, and ribosome rescue. The results of these studies will reveal the mechanisms that drive and regulate translation in eukaryotes, providing a framework for investigating the role of translational control in human health and disease.