eIF2A in translational control

真核生物蛋白质合成的启动是一个复杂的过程，需要12种以上不同的启动。 因子，由30多个多肽链组成。其中许多因子的作用已经确定。 然而，其中一些组织的确切作用及其作用机制仍然不是很好 明白了。EIF2A是一种65 kDa的单链蛋白，最初被认为是作为 原核生物IF2的同系物，因为eIF2A和IF2催化生化相似的反应，即它们刺激 启动子甲硫酰-tRNA(Met-tRNAMeti)与核糖体小亚基结合。然而，随后 对杂三聚体126 kDa因子eIF2()的鉴定表明，该因子而不是eIF2a主要是 负责真核生物中Met-tRNAMeti与40S核糖体亚基的结合。在哺乳动物中，有四种压力-- 激活的激酶通过使eif2亚单位磷酸化来降低活性eif2的水平，因此， 降低全球翻译水平。然而，许多细胞和病毒蛋白的翻译似乎是 尽管需要Met-tRNAMeti，但对eif2α磷酸化具有抵抗力。结果发现，因素的一个子集， 包括eIF2A在内，在一定条件下可以促进Met-tRNAMeti高效募集到40S/mRNA复合体 EIF2活性的抑制，或它的缺失。最近，有报道称eIF2a也参与了非8月 高等真核生物的依赖启动与主要组织相容性对抗原提呈的控制 复合体(MHC)I类分子，整合的应激反应和肿瘤的发生和发展。所有的 在细胞模型中，这些事件受到eIF2A沉默的影响。然而，eIF2A在体内的确切作用也是如此 因为它的确切作用机制在很大程度上仍然是个谜。我们有一个根本性的差距 了解eIF2A如何在体内和体外哺乳动物系统中发挥作用。为了填补上面的空白， 为了继续研究真核/哺乳动物eIF2A的物理和功能特征，我们创建了一个 活体纯合子eIF2A-全基因敲除小鼠株及重组eIF2A在大肠杆菌中的表达 细胞。这项建议的最终目的是了解eIF2A在体内和体外的功能。这一目标将 通过体外、体外和体内(小鼠模型)方法相结合的方法来实现。这样做的结果是 这将对理解基因翻译控制的基本机制具有重要意义 在高等真核生物中的表达，尤其是作为应激反应的一部分。

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action still remain not well understood. eIF2A is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e. they stimulate initiator methionyl-tRNA (Met-tRNAMeti) binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2() showed that this factor and not eIF2A is primarily responsible for the binding of Met-tRNAMeti to 40S ribosomal subunits in eukaryotes. In mammals, four stress- activated kinases reduce the level of active eIF2 by phosphorylating the eIF2subunit and, consequently, reducing the global level of translation. However, translation of many cellular and viral proteins appeared to be resistant to eIF2α phosphorylation despite requiring Met-tRNAMeti. It was found that a subset of factors, including eIF2A, can promote efficient recruitment of Met-tRNAMeti to 40S/mRNA complexes under conditions of inhibition of eIF2 activity, or its absence. Recently, eIF2A was also reported to be involved in non-AUG dependent initiation in higher eukaryotes and the control of antigen presentation by major histocompatibility complex (MHC) class I molecules, the integrated stress response and tumor initiation and progression. All of these events were affected by eIF2A silencing in cellular models. Yet, the precise role of eIF2A in vivo, as well as the precise mechanism of its action still remain largely enigmatic. There is a fundamental gap in our understanding of how eIF2A functions in mammalian systems in vivo and ex vivo. To fill in this gap above and to continue the physical and functional characterization of a eukaryotic/mammalian eIF2A, we have created a viable homozygous eIF2A-total knockout mouse strain and obtained recombinant eIF2A expressed in E. coli cells. The ultimate goal of this proposal is to understand the function of eIF2A in vivo and in vitro. This goal will be achieved by a combination of in vitro, ex vivo and in vivo (mouse model) approaches. The outcome of this proposal will be important for understanding the basic mechanisms of the translational control of gene expression in higher eukaryotes, especially as part of the stress response.