The structural dynamics of ribosomal frameshifting and ribosome rescue

项目摘要 核糖体将信使RNA（mRNA）翻译成蛋白质， 机器（TM）是基因表达的一个基本步骤，对生命至关重要。因为细菌TM是一种 已被证明是开发新抗生素的靶点，而且由于许多人类疾病都是由 与翻译失调有关，细菌中的翻译和翻译控制机制， 真核生物仍处于深入研究之中。在过去的二十年里，结构研究表明， TM在蛋白质合成过程中经历的大规模结构重排。不幸的是，尺寸， TM的复杂性和构象灵活性极大地阻碍了这些动力学的研究， 大大限制了我们对这些动态如何有助于翻译机制的理解， 翻译控制尽管如此，使用单分子荧光和结构荧光的组合， 技术，我们和其他人已经能够描述动态的核心步骤的翻译， 细菌TM尽管取得了这些成就，但我们对是否和 这些核心步骤的动力学是如何作为生物医学上重要的翻译控制的一部分进行调节的 战略布局为了填补这些空白，在本申请的第一个目的中，我们提出使用单- 分子荧光，结构和生物化学的方法来研究如何动态的 细菌TM被调节以驱动核糖体移码。框架转换是一种翻译控制 策略，其中TM向后或向前滑动mRNA上的一个或多个核苷酸， - 插入或缺失“移码”突变，其否则将导致产生异常或 所述方法可用于合成截短的蛋白质或驱动来自单个mRNA的多于一种蛋白质产物的合成。这些 实验有望揭示框架转换背后仍然难以捉摸的机制。第二个目标，我们 将使用类似的方法来研究核糖体拯救因子如何调节细胞的动力学。 细菌TM作为它们识别和拯救具有以下特征的核糖体的机制的一部分， 会受到威胁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。第三个目标， 我们将单分子荧光和结构技术的结合扩展到酵母翻译 系统，使我们能够调查真核生物的翻译，移码， 和核糖体拯救。这些研究的结果将揭示驱动和调节的机制 翻译在真核生物中的作用，提供了一个框架，研究翻译控制在人类 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.