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

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，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，而成年人需要更慢的生长速度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。几乎所有的细胞功能都是由蛋白质完成的。每一个都是由被称为氨基酸的积木组成的，这些氨基酸链接在一起并折叠成三维结构，这些结构对每个人履行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”，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。这一建议涉及启动阶段，在该阶段中，一组专门的因素发挥作用。蛋白质合成启动因子指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，并将其定位到每个mRNA上的正确起始点。这对于在每个细胞中制造正确的蛋白质是至关重要的。这必须准确和快速地完成。启动是蛋白质合成中最复杂的阶段，也是在分子水平上最不被理解的阶段。该提案涉及指定的因子eIF2B、eIF2和Met-tRNAi。在这项建议中，我们描述了已经发现了因子eIF2B的一种新功能的初步实验。EIF2B被认为是一种“启动”其伴侣eIF2的因子，使eIF2能够与Met-tRNAi结合，并将其招募到核糖体中。我们发现eIF2B还有第二个作用，可以从eIF2中去除另一个因素(EIF5)。这发生在eIF2B打开eIF2之前。EIF2B是一个由五个部分组成的复杂蛋白质，现有的和新的角色只需要其中的两个。遗传和生化研究，包括许多在我们实验室完成的研究，表明eIF2B还有更重要的作用，在这里我们概述了我们的研究计划。具体地说，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突变会导致一种称为“消失性白质病”(VWM)的致命脑部疾病，我们将研究导致VWM的突变如何改变这些新的活动。由于蛋白质合成是对我们所有细胞至关重要的几项功能之一，我们认为，在我们在这一研究领域处于领先地位的情况下，现在研究这一功能很重要。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VWM是如何导致疾病的。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。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.