Mechanisms of assembly and remodeling of human translation initiation complexes

描述(由申请人提供)：蛋白质合成的控制(翻译)对细胞的增殖和分化至关重要。在人类中，翻译的启动是一个多步骤的过程，涉及两个关键的GTP水解步骤。翻译起始依赖于超道尔顿翻译起始复合体中核糖体、RNA和蛋白质之间的动态相互作用网络。GTP酶的两个步骤尤其如此：开始密码子选择和核糖体亚基连接，在这两个步骤中，许多相互作用是合作的，而其他相互作用是相互竞争的。我们假设，启动复合体各组分之间的协同作用确保了它们被招募到复合体中，它们正确的定位，以及启动复合体作为一个整体的稳定性。我们认为，一对竞争的相互作用对起始复合体的重塑负责，通过该复合体的成熟，一组相互作用被另一组相互作用所取代，从而形成一个准备开始合成蛋白质的活性核糖体。为了验证这些假设，我们提出了一种多学科的创新方法，它将结合使用核磁共振、荧光光谱和其他生物物理和生物化学方法。1.我们将使用生物物理分析和核磁共振技术来确定负责起始密码子选择和核糖体亚基连接的蛋白质之间的哪些相互作用是协同的，哪些是竞争的，并从结构上表征所涉及的结合界面。2.通过对体外重组翻译起始复合体的稳态荧光各向异性、时间分辨荧光各向异性衰变和生物化学分析，阐明蛋白质在起始复合体中募集和释放的时间规律。3.我们将使用荧光共振能量转移(FRET)来确定负责起始密码子选择和核糖体亚基连接的蛋白质在起始复合体中的位置和相互方向。已知位置的蛋白质将作为参考。总之，这些目标的结果将提供对起始密码子选择和核糖体亚基连接的协调和调节的全面理解。这一建议的长期目标是阐明人类翻译起始复合体的结构组织，以及它们的组装和重塑机制。这项工作将确定翻译启动过程中的关键步骤，这些步骤是为了治疗目的而操纵蛋白质合成速率的有希望的目标。 公共卫生相关性： 积极分裂细胞，如癌细胞，需要增加蛋白质合成的速度，而蛋白质合成的抑制剂已被证明在癌症治疗中有效。这项提案中概述的工作将为直接和特定地抑制用于癌症治疗的蛋白质合成建立新的靶点。

DESCRIPTION (provided by applicant): Control of protein synthesis (translation) is vital for cell proliferation and differentiation. In human, initiation of translation is a multi-step process that involves two key GTP hydrolysis steps. Translation initiation relies on a dynamic network of interactions among ribosomes, RNAs and proteins within a mega-dalton translation initiation complex. This is particularly true for the two GTPase steps: start codon selection and ribosomal subunit joining, where a number of interactions are cooperative while others are competing with each other. We hypothesize that synergistic interactions among components of the initiation complex ensure their recruitment to the complex, their proper positioning, as well as the stability of the initiation complex as a whole. We propose that pairs of competing interactions are responsible for remodeling of the initiation complex, whereby one set of interactions are replaced by another set of interactions as the complex matures toward the formation of an active ribosome ready to start synthesizing protein. To test these hypotheses, we propose a multidisciplinary innovative approach, which will use a combination of NMR, fluorescence spectroscopy and other biophysical and biochemical methods. 1. We will use biophysical assays and NMR to determine which interactions among the proteins responsible for start codon selection and ribosomal subunit joining are cooperative and which are competitive and to characterize structurally the binding interfaces involved. 2. We will use steady-state fluorescence anisotropy, time-resolved fluorescence anisotropy decay and biochemical assays on in vitro reconstituted translation initiation complexes to elucidate the temporal regulation of the recruitment of proteins to the initiation complex and their release. 3. We will use Fluorescence Resonance Energy Transfer (FRET) to determine the positions and mutual orientations within the initiation complex of the proteins responsible for start codon selection and ribosomal subunit joining. Proteins with known positions will serve as reference. Together, the results from these aims will provide a comprehensive understanding of the coordination and regulation of start codon selection and ribosomal subunit joining. The long term goals of this proposal are to elucidate the structural organization of the human translation initiation complexes, as well as the mechanisms of their assembly and remodeling. This work will identify key steps in the translation initiation process that are promising targets in manipulating the rates of protein synthesis for therapeutic purposes. PUBLIC HEALTH RELEVANCE: Actively dividing cells, such as cancer cells, require increased rates of protein synthesis and inhibitors of protein synthesis have proved effective in cancer therapy. The work outlined in this proposal will establish new targets for direct and specific inhibition of protein synthesis for use in cancer therapy.