Regulators of Translation Elongation Factor eEF1A

描述（由申请人提供）：真核翻译延伸是蛋白质合成过程中保真度和持续合成能力的决定因素。这一过程的调控主要基于真核细胞延伸因子1A（eEF 1A）及其辅因子鸟嘌呤核苷酸交换因子eEF 1Ba、氨酰-tRNA（aa-tRNA）、真菌特异性延伸因子eEF 3和肌动蛋白细胞骨架之间的功能关系。eEF 1A是GTP结合蛋白，其将aa-tRNA递送至核糖体A位点。eEF 1A需要鸟嘌呤核苷酸交换因子（GEF）eEF 1Ba？用于重新激活为GTP结合形式。eEF 3是一种重要的真菌特异性延伸因子，与eEF 1A具有功能和物理相互作用。eEF 1A还与翻译装置外的蛋白质相互作用，特别是肌动蛋白和肌动蛋白结合蛋白。多个真核系统中的证据指出细胞骨架在翻译调节中的功能作用，其中eEF 1A-肌动蛋白相互作用可能起关键作用。该建议的统一思路是假设eEF 1A，其在蛋白质合成中的辅助因子（aa-tRNA，eEF 1Ba和eEF 3）和肌动蛋白之间的功能相互作用是蛋白质合成和细胞骨架之间的关键调节和通信点。eEF 1A在这些角色之间的分布将随后影响基因表达的调节。目的1是基于我们的假设，即eEF 1A是肌动蛋白捆绑蛋白家族的成员，其作用与其延伸活性无关，但通过肌动蛋白细胞骨架连接到翻译起始。目的2是基于我们的假设，除了作为鸟嘌呤核苷酸交换因子的作用，eEF 1Ba有助于促进aa-tRNA结合到GTP结合形式的eEF 1A，从而与肌动蛋白竞争结合到eEF 1A。目的3是基于我们关于eEF 3-eEF 1A相互作用的数据，该数据支持我们的模型，即eEF 3与eEF 1A的结合与肌动蛋白结合是互斥的。此外，eEF 3的晶体结构和与80 S酵母核糖体结合的cryo-EM重建导致以下假设：染色体结构域和ABC 2结构域对于eEF 3在核糖体上的功能及其对A位点处的翻译保真度的影响是必不可少的。总的来说，这项工作将作为延伸因子在人类系统中功能的模型，关于eEF 1A在协调翻译延伸和细胞骨架之间的相互作用方面的独特作用。此外，这将阐明在不同生理条件和疾病状态下发生的eEF 1A水平和活性改变的功能后果。此外，我们对真菌翻译延伸的更多了解可以作为未来靶向该过程的真菌特异性方面（如eEF 3）的平台，用于开发新的抗真菌治疗方法。对于酿酒酵母，先前资助期间的进展现在可以用于对eEF 1A及其相关因子在基因表达调控背景下的功能作用，物理状态和独特方面进行综合遗传，生物化学和细胞生物学分析。

DESCRIPTION (provided by applicant): Eukaryotic translation elongation is a determinant of fidelity and processivity during protein synthesis. The regulation of this process is largely based on the functional relationship between eukaryotic Elongation Factor 1A (eEF1A) and its cofactors the guanine nucleotide exchange factor eEF1Ba, aminoacyl-tRNA (aa-tRNA), the fungal specific elongation factor eEF3, and the actin cytoskeleton. eEF1A is the GTP binding protein that delivers aa-tRNA to the ribosomal A-site. eEF1A requires the guanine nucleotide exchange factor (GEF) eEF1Ba? for reactivation to the GTP-bound form. eEF3 is an essential fungal-specific elongation factor that has a functional and physical interaction with eEF1A. eEF1A further interacts with proteins outside the translational apparatus, in particular actin and actin binding proteins. Evidence in multiple eukaryotic systems points to a functional role of the cytoskeleton in translational regulation, where the eEF1A-actin interaction likely plays a key role. The unifying thread of this proposal is the hypothesis that the functional interplay between eEF1A, its co-factors in protein synthesis (aa-tRNA, eEF1Ba, and eEF3), and actin is a key regulatory and communication point between protein synthesis and the cytoskeleton. The distribution of eEF1A between these roles would subsequently affect the regulation of gene expression. Aim 1 is based on our hypothesis that eEF1A is a member of the actin bundling protein family that performs a role unrelated to its elongation activity but linked through the actin cytoskeleton to translation initiation. Aim 2 is based on our hypothesis that in addition to its role as a guanine nucleotide exchange factor, eEF1Ba helps facilitate aa-tRNA binding to the GTP-bound form of eEF1A and thus competes with actin for binding to eEF1A. Aim 3 is based on our data on the eEF3-eEF1A interaction that supports our model that eEF3 binding to eEF1A is mutually exclusive to actin binding. Furthermore, the crystal structure of eEF3 and cryo-EM reconstitution bound to the 80S yeast ribosome led to the hypothesis that the chromodomain and the ABC2 domain are essential for the function of eEF3 on the ribosome and its effects on translational fidelity at the A-site. Overall, the work will serve as a model for elongation factor function in human systems regarding the unique role of eEF1A in coordinating the interplay between translation elongation and the cytoskeleton. Further, this will shed light on the functional consequences of altered eEF1A levels and activities demonstrated to occur in different physiological conditions and disease states. In addition, our increased knowledge of fungal translation elongation can serve as a platform for the future targeting of the fungal-specific aspects of this process, such as eEF3, for the development of new antifungal treatments. With the yeast Saccharomyces cerevisiae, the progress in the prior funding period can now be utilized for an integrated genetic, biochemical and cell biological analysis on the functional roles, physical states and unique aspects of eEF1A and its associated factors in the context of regulated of gene expression.