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

所有生物都是由细胞组成的。细胞生长和细胞分裂是由一系列信号协调和控制的，这些信号确保它们在正确的地点和时间发生。因此，发育中的胚胎需要一段快速生长的时期，而成年人需要缓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，可能会发生癌症等疾病，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。每种蛋白质都是由称为氨基酸的构建模块组成的，氨基酸以链连接并折叠以形成三维结构，这些结构对于每个蛋白质履行其各自的角色都很重要。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”，它是称为“基因表达”的途径的最后一步，这对于确保正确的基因在正确的地点和时间被解码至关重要。蛋白质合成发生在称为核糖体的分子机器内，核糖体解码从称为信使RNA（mRNA）的中间分子内的基因组传递的指令。每个人类细胞都含有超过一百万个核糖体。核糖体对mRNA的解码是通过称为蛋白质合成因子的“助手”和称为转移RNA（tRNA）的衔接分子的协同作用实现的，这些衔接分子将必需的氨基酸结合在一起。这个提议涉及蛋白质合成的起始阶段，其中一组专门的因子起作用，称为蛋白质合成起始因子。它们将核糖体和一种称为起始tRNA的特殊tRNA引导到每个mRNA上的正确起始位置，该起始tRNA以氨基酸甲硫氨酸（指定为Met-tRNAi）启动蛋白质。这对于在每个细胞中制造正确的蛋白质至关重要。这必须准确而迅速地完成。起始是蛋白质合成中最复杂的阶段，也是分子水平上最不清楚的阶段。该提议涉及命名为eIF 2B和eIF 2的因子，这两个因子对于调节Met-tRNAi的核糖体结合至关重要。eIF 2B被认为是一个因子，它可以“打开”它的伴侣eIF 2，使eIF 2可以结合到Met-tRNAi并将其招募到核糖体中。在这个提案中，我们描述了初步实验，表明因子eIF 2B是以前认为的两倍大，也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，因此我们建议使用称为冷冻电子显微镜的技术，将高度纯化的蛋白质样品在液氮中快速冷冻，然后将其放大，以便我们可以构建eIF 2B的三维模型，并研究它如何与其伴侣蛋白eIF 2结合。 通过详细了解eIF 2B对蛋白质合成控制的贡献，将有助于了解细胞生长的控制，并进一步了解由eIF 2B突变引起的消失性白色疾病。这项工作也可能是感兴趣的行业，例如那些生产特定的蛋白质作为药物治疗或商业产品或那些通过发酵生长细胞，因为它将允许一个更好的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，将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.