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时间和地点发生。因此，发育中的胚胎需要一段时间的快速生长，而成人需要更慢的生长来替换受损或死亡的细胞。当失去对生长的控制时，癌症等疾病就会发生，而当需要时不能促进生长可能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将营养物质转化为生命所需的新蛋白质。蛋白质几乎执行所有的细胞功能。每种蛋白质都是由被称为氨基酸的构建块组成的，氨基酸以链连接并折叠成三维结构，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是由基因组中基因的DNA序列决定的。这被称为“蛋白质合成”，它是“基因表达”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中，核糖体解码来自基因组的指令，这些指令传递在称为信使rna （mrna）的中间分子中。每个人体细胞含有超过一百万个核糖体。核糖体对mRNA的解码是通过蛋白质合成因子和转运rna （trna）分子的协同作用实现的，转运rna将必需的氨基酸聚集在一起。该建议涉及蛋白质合成的起始阶段，其中一组专用因子起作用，称为蛋白质合成起始因子。它们引导核糖体和一种特殊的tRNA，称为启动器tRNA，将含有氨基酸蛋氨酸的蛋白质（称为Met-tRNAi）启动到每个mRNA上的正确起始位置。这对于在每个细胞中制造正确的蛋白质至关重要。这件事必须既准确又迅速。起始是蛋白质合成过程中最复杂的阶段，也是分子水平上了解最少的阶段。该建议涉及eIF2B和eIF2因子，这两个因子对调节Met-tRNAi的核糖体结合至关重要。eIF2B是一种被称为“开启”其伴侣eIF2的因子，因此eIF2可以与Met-tRNAi结合并将其招募到核糖体中。在本提案中，我们描述了初步实验，这些实验表明，因子eIF2B是之前认为的两倍大，并且还提供了其三维整体结构的线索。由于蛋白质无法用肉眼或光学显微镜看到，因此我们建议使用冷冻电子显微镜技术将高度纯化的蛋白质样品快速冷冻在液氮中，然后将其放大，这样我们就可以建立eIF2B的三维模型，并研究它如何与它的伴侣蛋白eIF2结合。通过详细了解eIF2B对蛋白质合成控制的贡献，将有助于理解细胞生长的控制，并进一步了解由eIF2B突变引起的白质消失病。这项工作也可能对那些生产特定蛋白质作为药物治疗或商业产品的行业或那些通过发酵培养细胞的行业感兴趣，因为它将使人们更好地了解蛋白质合成机制。详细的结构信息将有助于揭示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.