Structural Dynamics of Translation

摘要 核糖体沿着mRNA运动是所有生物体中蛋白质合成的基本方面。期间 翻译延伸核糖体以逐个密码子的方式沿着mRNA移动并解开mRNA 二级结构然而，特定的mRNA序列诱导核糖体停滞和移码。我们的目标 为了理解为什么这些特定的RNA序列减慢了核糖体的移位，而核糖体 在大多数mRNA结构中没有长时间的停顿。利用单分子荧光 显微镜和体外翻译系统组装纯化的成分，我们将定义的性质， 移码诱导RNA茎环和Shine-Dalgarno样序列，这些序列对核糖体至关重要 暂停。阐明RNA结构在翻译延伸调节中的作用是研究RNA翻译的关键。 理解核糖体移码的分子机制，这对合成 许多细菌、真核生物和病毒蛋白质。例如，生产蛋白酶，逆转 人类免疫缺陷病毒（HIV）的转录酶和整合酶依赖于一个-1程序化的核糖体 移码事件此外，核糖体停顿是许多共翻译的主要决定因素， 包括蛋白质加工、折叠和靶向膜的过程。翻译的转调 延伸率还影响mRNA的稳定性并调节所产生的蛋白质的水平。 除了密码子-密码子易位，另一种核糖体运动称为核糖体扫描， 发生在真核生物蛋白质合成的起始阶段。据信，在真核生物期间， 在翻译起始时，核糖体小亚基首先被募集到mRNA的5'末端，然后扫描5'末端， 起始密码子的mRNA的非翻译区（5 'UTR）。扫描的分子机制， 对于正确的起始密码子选择至关重要的是知之甚少。此外，实时运动的 未检测到沿5 'UTR的小核糖体亚基沿着。我们将使用单分子荧光 设计用于检测核糖体小亚基的运动并测量核糖体小亚基的运动速率的显微镜分析。 扫描。我们将研究核糖体扫描的机制，并获得对真核生物的重要见解。 翻译起始，其失调与许多人类疾病有关，包括癌症和 糖尿病 总之，拟议的研究将大大有助于建立分子机制 蛋白质的合成，并为未来的抗病毒和癌症治疗的发展提供了基础。

Abstract Ribosomal movement along mRNA is an essential facet of protein synthesis in all organisms. During translation elongation the ribosome moves along mRNA in a codon-by-codon manner and unwinds mRNA secondary structure. However, specific mRNA sequences induce ribosome stalling and frameshifting. We aim to understand why these specific RNA sequences slow down ribosome translocation while the ribosome translocates through most mRNA structure without long pauses. Using single-molecule fluorescence microscopy and an in vitro translation system assembled from purified components, we will define properties of frameshift-inducing RNA stem-loops and Shine-Dalgarno-like sequences that are critical for the ribosome pausing. Elucidating the role of RNA structure in the modulation of translation elongation is a key to understanding the molecular mechanism of ribosome frameshifting, which is essential for the synthesis of numerous bacterial, eukaryotic and viral proteins. For example, the production of protease, reverse transcriptase and integrase of human immunodeficiency virus (HIV) depends on a -1 programmed ribosomal frameshifting event. Furthermore, ribosome pausing is a major determinant of numerous co-translational processes including protein processing, folding and targeting to membranes. Modulation of the translation elongation rate also affects mRNA stability and regulates levels of the produced protein. In addition to codon-by-codon translocation, another type of ribosome movement termed ribosome scanning occurs during the initiation phase of protein synthesis in eukaryotes. It is believed that during eukaryotic translation initiation the small ribosomal subunit is first recruited to the 5' end of mRNA and then scans the 5'- untranslated region (5'UTR) of the mRNA for the start codon. The molecular mechanism of scanning, which is critical for correct start codon selection, is poorly understood. Furthermore, the real-time movement of the small ribosomal subunit along the 5'UTR was never detected. We will use a single-molecule fluorescence microscopy assay designed to detect the movement of the small ribosomal subunit and measure the rate of the scanning. We will examine the mechanism of ribosome scanning and gain critical insights into eukaryotic translation initiation, deregulation of which is linked to a number of human diseases including cancer and diabetes. Taken together, the proposed studies will substantially contribute to establishing the molecular mechanisms of protein synthesis and provide a basis for the future development of antiviral and cancer therapies.