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

真核蛋白质合成因子复合物 eIF2B 和 eIF2/eIF2B 的结构研究，对真核细胞的翻译控制至关重要

基本信息

批准号：
BB/L020157/1
负责人：
Graham Pavitt
金额：
$ 48.29万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL020157%2F1
关键词：
Structural studies eukaryotic protein synthesis

项目摘要

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.

所有生物体都是由细胞组成的。细胞生长和细胞分裂由多种信号协调和控制，确保它们在正确的地点和时间发生。因此，发育中的胚胎需要快速生长的时期，而成年人则需要慢得多的生长来替换受损或垂死的细胞。当生长失去控制时，就会出现癌症等疾病，而在需要时未能促进生长可能会导致受损细胞无法修复或导致组织浪费。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。每种蛋白质均由称为氨基酸的结构单元组成，这些结构单元以链连接并折叠以形成 3 维结构，这对于每个蛋白质履行其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”，它是“基因表达”途径的最后一步，这对于确保在正确的地点和时间解码正确的基因至关重要。蛋白质合成发生在称为核糖体的分子机器内，该分子机器解码从称为信使 RNA (mRNA) 的中间分子中的基因组传递的指令。每个人体细胞含有超过一百万个核糖体。核糖体对 mRNA 的解码是通过称为蛋白质合成因子的“辅助因子”和称为转移 RNA (tRNA) 的接头分子的协同作用而实现的，这些接头分子将必需的氨基酸聚集在一起。该提案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。他们将核糖体和一种称为起始 tRNA 的特殊 tRNA 引导至每个 mRNA 上的正确起始位置，该 tRNA 将带有氨基酸蛋氨酸（称为 Met-tRNAi）的蛋白质启动。这对于在每个细胞中制造正确的蛋白质至关重要。这必须准确且快速地完成。起始阶段是蛋白质合成中最复杂的阶段，也是分子水平上最难理解的阶段。该提案涉及 eIF2B 和 eIF2 因子，这两个因子对于调节 Met-tRNAi 的核糖体结合至关重要。 eIF2B 被称为“打开”其伙伴 eIF2 的因子，以便 eIF2 可以与 Met-tRNAi 结合并将其招募到核糖体。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，并且还提供了其三个维度整体结构的线索。由于肉眼或光学显微镜无法看到蛋白质，因此我们在此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，以便我们可以构建 eIF2B 的三维模型并研究它如何与其伴侣蛋白 eIF2 结合。通过详细了解 eIF2B 对蛋白质合成控制的贡献，将有助于了解细胞生长的控制，并进一步深入了解由 eIF2B 突变引起的白质消失疾病。这项工作也可能引起行业的兴趣，例如那些生产特定蛋白质作为药物治疗或商业产品的行业或那些通过发酵培养细胞的行业，因为它将允许更好地理解蛋白质合成机制。详细的结构信息将有助于揭示蛋白质合成的精确控制，将有助于设计优化的商业蛋白质表达或发酵系统。