Investigating novel steps for promoting tRNA binding to translation factor eIF2 during protein synthesis initiation

所有生物都是由细胞组成的。细胞生长和分裂是由广泛的信号协调和控制的，这些信号确保它们只在适当的时间发生。在极端情况下，发育中的胚胎需要快速生长，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。几乎所有的细胞功能都是由蛋白质完成的。每一种都是由一种叫做氨基酸的构建块组成的，氨基酸以链的形式连接并折叠成三维结构，这对每一种氨基酸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。该提案涉及启动阶段，其中一组专用因素起作用。蛋白质合成起始因子指导核糖体和一种特殊的tRNA，称为启动器tRNA，它将蛋白质与氨基酸蛋氨酸（称为Met-tRNAi）连接到每个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。起始阶段是蛋白质合成过程中最复杂的阶段，也是在分子水平上理解得最少的阶段。本提案涉及指定为eIF2B、eIF2和Met-tRNAi的因子。在本提案中，我们描述了初步实验，发现了因子eIF2B的新功能。eIF2B被认为是一种“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。我们发现eIF2B具有从eIF2中去除另一个因子（eIF5）的第二个作用。这发生在eIF2B开启eIF2之前。eIF2B是一个由五个部分组成的复杂蛋白，现有的和新的作用只需要其中的两个部分。遗传和生化研究，包括在我们实验室完成的许多研究，表明eIF2B具有进一步的重要作用，在这里我们概述了我们的研究计划。具体来说，我们将评估eIF2B如何加速Met-tRNAi与eIF2的结合，以及Met-tRNAi如何促进eIF2B的去除。由于eIF2B突变导致致命的脑部疾病“消失性白质病”（VWM），我们将研究导致VWM的突变如何改变这些新的活动。因为蛋白质合成是我们所有细胞的几个关键功能之一，我们认为现在研究这一点很重要，虽然我们在这一研究领域处于领先地位。通过详细了解eIF2B对蛋白质合成控制的贡献，它将有助于理解细胞生长的控制，并进一步了解VWM如何引起疾病。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。通过了解蛋白质合成的精确控制，这可能有助于设计优化的商业蛋白质表达或发酵系统。

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 only at appropriate times. At the extremes, developing embryos require 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. Almost all cellular functions are performed by proteins. Each one 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 in which a dedicated set of factors act.. Protein synthesis initiation factors 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, eIF2 and Met-tRNAi. In this proposal we describe preliminary experiments that have uncovered a novel function for the factor eIF2B. 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. We have found that eIF2B has a second role to remove another factor (eIF5) from eIF2. This happens before eIF2B switches on eIF2. eIF2B is a complicated protein with five parts and both the existing and new roles only require 2 of them. Genetic and biochemical studies, including many done in our laboratory, indicate that eIF2B has further essential roles and here we outline our plan to investigate those. Specifically we will evaluate how eIF2B accelerates Met-tRNAi binding to eIF2 and how Met-tRNAi then promotes removal of eIF2B. As eIF2B mutations cause the fatal brain disease called 'Vanishing white matter disease' (VWM) we will investigate how the mutations causing VWM alter these new activities. Because protein synthesis is one of several functions critical for all our cells we believe that it is important to study this now, while we have a lead in this area of research. 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 how VWM causes disease. 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. By understanding the precise controls of protein synthesis this may help in the design optimized commercial protein expression or fermentation systems.