Mechanisms of assembly and remodeling of human translation initiation complexes

描述（由申请人提供）：蛋白质合成（翻译）的控制对于细胞增殖和分化至关重要。在人类中，翻译的起始是一个多步骤的过程，涉及两个关键的GTP水解步骤。翻译起始依赖于一个巨道尔顿翻译起始复合物中核糖体、RNA和蛋白质之间相互作用的动态网络。这对于两个GTTT步骤尤其如此：起始密码子选择和核糖体亚基连接，其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，协同作用的起始复合物的组件之间的相互作用，确保他们的招聘复杂，其正确的定位，以及作为一个整体的稳定性的起始复合物。我们提出，对竞争的相互作用是负责重塑的起始复合物，其中一组相互作用被取代的另一组相互作用的复杂成熟的形成一个活跃的核糖体准备开始合成蛋白质。 为了验证这些假设，我们提出了一种多学科的创新方法，该方法将使用NMR，荧光光谱和其他生物物理和生物化学方法的组合。1.我们将使用生物物理测定和NMR来确定哪些蛋白质之间的相互作用负责起始密码子选择和核糖体亚基连接是合作的，哪些是竞争性的，并在结构上表征所涉及的结合界面。2.我们将使用稳态荧光各向异性，时间分辨荧光各向异性衰减和生化分析在体外重建的翻译起始复合物，以阐明的时间调节的蛋白质的招聘起始复合物和它们的释放。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的起始复合物内的位置和相互取向。具有已知位置的蛋白质将用作参考。 总之，从这些目标的结果将提供一个全面的理解起始密码子的选择和核糖体亚基连接的协调和调节。该计划的长期目标是阐明人类翻译起始复合物的结构组织，以及它们的组装和重塑机制。这项工作将确定翻译起始过程中的关键步骤，这些步骤是操纵蛋白质合成速率以达到治疗目的的有希望的目标。 公共卫生相关性： 活跃分裂的细胞，如癌细胞，需要增加蛋白质合成的速率，并且蛋白质合成的抑制剂已被证明在癌症治疗中有效。本提案中概述的工作将建立用于癌症治疗的直接和特异性抑制蛋白质合成的新靶点。

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.