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

研究蛋白质合成起始过程中促进 tRNA 与翻译因子 eIF2 结合的新步骤

基本信息

批准号：
BB/L000652/1
负责人：
Graham Pavitt
金额：
$ 39.86万
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：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL000652%2F1
关键词：
Investigating novel steps promoting tRNA

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 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.

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。几乎所有的细胞功能都是由蛋白质完成的。每一个都是由称为氨基酸的构建模块组成的，这些氨基酸以链的形式连接并折叠，形成三维结构，这些结构对于每个人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。这一建议涉及启动阶段，在这一阶段有一套专门的因素发挥作用。蛋白质合成起始因子将核糖体和称为起始tRNA的专门tRNA引导到每个mRNA上的正确起始位置，起始tRNA将具有氨基酸甲硫氨酸的蛋白质（指定为Met-tRNAi）起始。这对于在每个细胞中制造正确的蛋白质至关重要。起始是蛋白质合成中最复杂的阶段，也是在分子水平上了解最少的阶段。该提议涉及命名为eIF 2B、eIF 2和Met-tRNAi的因子。在这个建议中，我们描述了初步的实验，发现了一个新的功能因子eIF 2B。 eIF 2B被认为是一种“打开”其配偶体eIF 2的因子，使得eIF 2可以与Met-tRNAi结合并将其招募到核糖体。我们已经发现eIF 2B具有从eIF 2去除另一个因子（eIF 5）的第二个作用。这发生在eIF 2B切换到eIF 2之前。eIF 2B是一个复杂的蛋白质，有五个部分，现有的和新的角色只需要其中的两个。遗传和生物化学研究，包括我们实验室进行的许多研究，表明eIF 2B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。具体而言，我们将评估eIF 2B如何加速Met-tRNAi与eIF 2的结合，以及Met-tRNAi如何促进eIF 2B的去除。由于eIF 2B突变导致致命的脑部疾病，称为“消失的白色物质疾病”（VWM），我们将研究如何突变导致VWM改变这些新的活动。由于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，因为我们在这一研究领域处于领先地位。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VWM如何导致疾病。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。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。