The protein synthesis production line: mechanisms and stress responses governing 'just-in-time' tRNA delivery to the ribosome

通过细胞tRNAs将氨基酸运送到核糖体是每个细胞蛋白质合成过程的核心，因此也是将细胞用作生物技术中生产蛋白质的‘工厂’的核心。在真核生物中，tRNA的传递是由一种需要GTP的翻译因子eEF1来执行的，eEF1是细胞中含量最丰富的蛋白质之一。EEF1拾取带氨基酸的tRNA，并将它们运送到翻译的核糖体，伴随着相关的GTP水解。然后，eEF1上的GDP被另外两个称为eEF1-β和伽马的延伸因子交换为GTP，允许循环再次开始。eEF1蛋白是至关重要的；未能迅速将tRNA运送到核糖体会导致核糖体在mRNA上的核糖体停滞，以及核糖体质量控制(RQC)途径的组件结合，从而触发核糖体放弃翻译。这个项目将描述我们的实验室已经发现的解决核糖体的新途径，这些核糖体由于eEF1缓慢的tRNA传递而停滞不前。我们将使用已建立的RQC应激反应分析，以及非途径翻译事件，如氨基酸错误掺入或核糖体移码，以探索核糖体如何在“空的核糖体受体位点”的情况下做出反应。其目的是确定新的核糖体应激反应途径，介导细胞对tRNA传递失败的反应。了解细胞如何在应对环境或生物技术挑战的同时维持氨基酸-tRNA传递到核糖体(并避免压力)，是我们面临的两个最重要的工业和医学挑战的关键：优化蛋白质的生物技术表达，以及了解这些过程受到影响的人类神经发育疾病。例如，生物技术使用基因表达来生产疫苗、药品和化学原料，但对宿主生物体来说，由此带来的生理挑战是相当大的，因为蛋白质合成是细胞中最耗能的过程之一，产量往往相应地受到影响。为了优化重组蛋白的表达，我们必须在分子水平上了解翻译需求的管理。博士生将使用先进的分子生物学方法，包括在酵母(阿伯丁实验室)和哺乳动物细胞(爱丁堡实验室)中对eEF1-tRNA递送系统及其相关的eEF1-β和伽马GTP循环装置进行基因组编辑，以了解EEF延长因子家族的活性如何在生物技术、健康和疾病中优化蛋白质合成。这项研究项目还将使用合成生物学和系统生物学建模相结合的方法，以了解tRNA是如何传递到核糖体的，以及当生物技术表达外源蛋白质对系统造成过度需求时引发的应激反应。该合成电路将用于开发一种生物技术适用的新型反馈电路，该电路可以感知导致核糖体受体空位的翻译压力，并通过降低蛋白质合成速率来做出反应，实现对蛋白质合成的自我控制，从而实时减少失速。将提供分子生物学、系统和合成生物学各方面的全面培训。

The delivery of amino acids to the ribosome by the cellular tRNAs is central to the process of protein synthesis in every cell, and therefore to the use of the cell as a protein-producing 'factory' in biotechnology. In eukaryotes tRNA delivery is carried out by a GTP-requiring translation factor called eEF1, one of the most abundant proteins in the cell. eEF1 picks up amino-acid charged tRNAs and delivers them to the translating ribosome, with associated GTP hydrolysis. GDP on eEF1 is then exchanged for GTP by two other elongation factors called eEF1-beta and gamma, allowing the cycle to begin again.The eEF1 proteins are crucially important; failure to deliver tRNA rapidly to the ribosome causes ribosome stalling on the mRNA, and binding of components of a ribosome quality control (RQC) pathway that triggers translation abandonment by the ribosome. This project will characterise new pathways that our lab has identified that resolve ribosomes stalled because of slow tRNA delivery by eEF1. We will use established assays for RQC stress responses, and off-pathway translation events such as misincorporation of amino acids or ribosomal frameshifting, to explore how the ribosome responds in 'empty ribosomal acceptor site' situations. The objective is to define the novel ribosomal stress response pathways that mediate cellular response to failed tRNA delivery. Understanding how cells maintain amino acid-tRNA delivery to the ribosome (and avoid stress) while responding to environmental or biotechnological challenges is key to two of our most important industrial and medical challenges: optimising biotechnological expression of proteins and understanding human neurodevelopmental disease in which these processes are compromised. Biotechnology, for instance, uses gene expression to produce vaccines, pharmaceuticals and chemical feedstocks, but the resulting physiological challenge for a host organism is considerable because protein synthesis is one of the most energetically demanding processes in the cell, and production yields are often correspondingly compromised. To optimise recombinant protein expression, we must understand the management of demand on translation at the molecular level.The PhD student will use advanced molecular biological methods including genome editing of the eEF1-tRNA delivery system and its associated eEF1-beta and gamma GTP recycling apparatus in yeast (Aberdeen lab) and mammalian cells (Edinburgh lab) to understand how the activity of this family of eEF elongation factors function to optimise protein synthesis in biotechnology, health and disease. This research project will also use a combination of synthetic biology and systems biology modelling, to understand how tRNAs are delivered to the ribosome, and the stress responses invoked when biotechnological expression of foreign proteins places excess demand on the systems. The synthetic circuit will be used to develop a biotechnologically-applicable novel feedback circuit that senses translational stress leading to vacant ribosome acceptor sites, and respond by reducing the protein synthetic rate to implement autogenous control of protein synthesis, thus reducing stalling in real-time. Full training in all aspects of molecular biology, systems and synthetic biology will be provided.