Regulators of Translation Elongation Factor eEF1A

描述（由申请人提供）：真核生物翻译伸长率是蛋白质合成过程中保真度和加工性的决定因素。这一过程的调控主要基于真核延伸因子1A （eEF1A）及其辅助因子鸟嘌呤核苷酸交换因子eEF1Ba、氨基酰基trna （aa-tRNA）、真菌特异性延伸因子eEF3和肌动蛋白细胞骨架之间的功能关系。eEF1A是GTP结合蛋白，将aa-tRNA传递到核糖体a位点。eEF1A需要鸟嘌呤核苷酸交换因子（GEF） eEF1Ba？以重新激活gtp绑定表单。eEF3是一种重要的真菌特异性延伸因子，与eEF1A具有功能和物理相互作用。eEF1A进一步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。多个真核系统的证据表明细胞骨架在翻译调节中的功能作用，其中eEF1A-actin相互作用可能起关键作用。该建议的统一思路是假设eEF1A及其在蛋白质合成中的辅助因子（aa-tRNA， eEF1Ba和eEF3）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。eEF1A在这些角色之间的分布随后会影响基因表达的调控。Aim 1是基于我们的假设，即eEF1A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与翻译起始有关。Aim 2基于我们的假设，即除了作为鸟嘌呤核苷酸交换因子的作用外，eEF1Ba还有助于促进aa-tRNA与gtp结合形式的eEF1A结合，从而与肌动蛋白竞争与eEF1A的结合。Aim 3基于我们关于eEF3-eEF1A相互作用的数据，该数据支持我们的模型，即eEF3与eEF1A的结合与肌动蛋白结合是互斥的。此外，eEF3的晶体结构和与80S酵母核糖体结合的冷冻电镜重构使我们假设，色域和ABC2结构域对于eEF3在核糖体上的功能及其对a位点翻译保真度的影响至关重要。总的来说，这项工作将作为人类系统中延伸因子功能的模型，关于eEF1A在协调翻译延伸和细胞骨架之间相互作用中的独特作用。此外，这将阐明在不同生理条件和疾病状态下发生的eEF1A水平和活动改变的功能后果。此外，我们对真菌翻译延伸的进一步了解可以作为未来针对该过程中真菌特异性方面的平台，例如eEF3，用于开发新的抗真菌治疗方法。通过对酿酒酵母的研究，前期资助期的进展可以用于对eEF1A及其相关因子在基因表达调控中的功能作用、物理状态和独特方面进行综合的遗传、生化和细胞生物学分析。

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.