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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，确保它们仅在适当的时间发生。在极端情况下，发育中的胚胎需要快速生长，而成年人则需要更慢的生长才能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。几乎所有的细胞功能都是由蛋白质执行的。每一种氨基酸都是由氨基酸组成，这些氨基酸以链的形式连接并折叠形成 3 维结构，这对于每个氨基酸履行其各自的作用非常重要。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。该提议涉及一组专用因子发挥作用的起始阶段。蛋白质合成起始因子引导核糖体和一种称为起始 tRNA 的特殊 tRNA，该 tRNA 将带有氨基酸蛋氨酸（称为 Met-tRNAi）的蛋白质启动到每个 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。该提案涉及指定为 eIF2B、eIF2 和 Met-tRNAi 的因子。在本提案中，我们描述了发现 eIF2B 因子新功能的初步实验。 eIF2B 被认为是“打开”其伙伴 eIF2 的因子，以便 eIF2 可以与 Met-tRNAi 结合并将其募集到核糖体上。我们发现 eIF2B 的第二个作用是从 eIF2 中去除另一个因子 (eIF5)。这发生在 eIF2B 打开 eIF2 之前。 eIF2B 是一种复杂的蛋白质，由五个部分组成，现有的和新的角色只需要其中的两个。遗传和生化研究（包括我们实验室进行的许多研究）表明 eIF2B 具有进一步的重要作用，在这里我们概述了研究这些研究的计划。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 突变会导致称为“白质消失病”(VWM) 的致命脑部疾病，因此我们将研究导致 VWM 的突变如何改变这些新活动。由于蛋白质合成是对我们所有细胞至关重要的多种功能之一，我们认为现在研究这一点很重要，而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，将有助于了解细胞生长的控制，并进一步深入了解 VWM 如何引起疾病。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。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。