Structural studies of eukaryotic protein synthesis factor complexes eIF2B and eIF2/eIF2B, critical for translational control in eukaryotic cells

所有的有机体都是由细胞组成的。细胞生长和细胞分裂受到一系列信号的协调和控制，确保它们发生在正确的地点和时间。因此，发育中的胚胎需要快速生长，而成人则需要慢得多的生长来取代受损或死亡的细胞。当失去生长控制时，可能会出现癌症等疾病，而在需要时未能促进生长可能会导致无法修复受损的细胞或导致组织浪费。我们一直在研究细胞如何控制营养物质转化为生命所需的新蛋白质。蛋白质执行几乎所有的细胞功能。每种蛋白质都是由被称为氨基酸的积木组成的，这些氨基酸以链的形式连接并折叠形成三维结构，这对每种蛋白质发挥各自的作用都很重要。正确制造每种蛋白质所需的指令由基因组中基因的DNA序列决定。这被称为“蛋白质合成”，是被称为“基因表达”的途径的最后一步，对于确保在正确的地点和时间破译正确的基因至关重要。蛋白质合成发生在被称为核糖体的分子机器中，这些机器解码被称为信使RNA(MRNAs)的中间分子内从基因组传递的指令。每个人类细胞都含有100多万个核糖体。核糖体对mRNA的解码是通过称为蛋白质合成因子的辅助分子和称为转移RNAs(TRNAs)的适配分子的协同作用来实现的，tRNAs将必要的氨基酸聚集在一起。这一建议涉及蛋白质合成的起始阶段，在该阶段中有一组专门的因子发挥作用，称为蛋白质合成起始因子。它们引导核糖体和一种称为启动子tRNA的特殊tRNA，该tRNA以氨基酸蛋氨酸(指定为Met-tRNAi)启动蛋白质，并将其连接到每一个mRNA的正确起始处。这对于在每个细胞中制造正确的蛋白质是至关重要的。这必须既准确又迅速地完成。起始是蛋白质合成过程中最复杂的阶段，也是在分子水平上最不被理解的阶段。这项建议涉及被指定为eIF2B和eIF2的因子，这两个因子对调节Met-tRNAi的核糖体结合至关重要。EIF2B被认为是一种因子，它能“启动”其伴侣eIF2，使eIF2能够与Met-tRNAi结合并将其招募到核糖体中。在这项提议中，我们描述了初步实验，这些实验表明eIF2B因子比之前认为的要大一倍，并提供了其三维整体结构的线索。由于肉眼或光学显微镜无法看到蛋白质，我们建议在这里使用一种名为低温电子显微镜的技术，在液氮中快速冷冻高度纯化的蛋白质样本，然后放大它们，以便我们可以建立eIF2B的三维模型，并研究它如何与其被称为eIF2的伙伴蛋白质结合。通过详细了解eIF2B对控制蛋白质合成的贡献，它将有助于了解细胞生长的控制，并进一步深入了解由eIF2B突变引起的消失的白质疾病。这项工作也可能引起工业的兴趣，例如那些生产特定蛋白质作为药物治疗或商业产品的工业，或那些通过发酵培养细胞的工业，因为它将有助于更好地理解蛋白质合成机制。详细的结构信息将有助于解开蛋白质合成的精确控制，有助于优化商业蛋白质表达或发酵系统的设计。

All organisms are composed of cells. Cell growth and cell division are coordinated and controlled by a wide range of signals that ensure they occur at the correct places and times. Hence developing embryos require periods of rapid growth while adults require much slower growth to replace damaged or dying cells. When there is a loss of growth control, diseases such as cancer can develop, while a failure to promote growth when required can cause a failure to repair damaged cells or cause tissue wasting. We have been studying how cells control the conversion of nutrients into the new proteins that are required for life. Proteins perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make 3-dimensional structures that are important for each to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of the genes in the genome. This is termed 'protein synthesis' and it is the final step in the pathway called 'gene expression' which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers' called protein synthesis factors and adapter molecules called transfer RNAs (tRNAs) that bring the necessary amino acids together. This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act that are called protein synthesis initiation factors. They direct the ribosome and a specialised tRNA called initiator tRNA that starts proteins with the amino acid methionine (designated Met-tRNAi) to the correct start place on each mRNA. This is critical to make the right proteins in every cell. This must be done both accurately and rapidly. Initiation is the most complex phase of protein synthesis and the least well understood at the molecular level. This proposal concerns factors designated eIF2B and eIF2, two factors critical for regulating ribosome binding of Met-tRNAi. eIF2B is known as a factor that 'switches on' its partner eIF2 so that eIF2 can bind to Met-tRNAi and recruit it to ribosomes In this proposal we describe preliminary experiments, which show that the factor eIF2B is twice as big as was previously thought and also provide clues to its overall structure in three dimensions. Because proteins cannot be seen with the naked eye or light microscopes, we propose here to use the technique called cryo electron microscopy to rapidly freeze highly purified protein samples in liquid nitrogen and then magnify them so that we can build a three-dimensional model of eIF2B and study how it binds to its partner protein called eIF2. By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help understand control of cell growth and provide further insight into vanishing white matter disease, which is caused by mutations in eIF2B. The work may also be of interest to industries eg those that produce specific proteins as drug therapeutics or for commercial products or those that grow cells by fermentation because it will allow an improved understanding of protein synthesis mechanism. Detailed structural information will help unravel the precise controls of protein synthesis will assist in the design of optimized commercial protein expression or fermentation systems.