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

通过细胞trna将氨基酸传递到核糖体是每个细胞中蛋白质合成过程的核心，因此也是将细胞用作生物技术中蛋白质生产“工厂”的关键。在真核生物中，tRNA的传递是由一种叫做eEF1的需要gtp的翻译因子来完成的，eEF1是细胞中最丰富的蛋白质之一。eEF1拾取带氨基酸的trna并将其传递给翻译核糖体，并伴随GTP水解。然后，eEF1上的GDP通过另外两个被称为eEF1- β和γ的延伸因子来交换GTP，从而使循环再次开始。eEF1蛋白至关重要；tRNA不能快速传递到核糖体，导致核糖体在mRNA上停滞，并结合核糖体质量控制（RQC）途径的成分，触发核糖体放弃翻译。该项目将描述我们实验室已经确定的解决由于eEF1递送tRNA缓慢而停滞的核糖体的新途径。我们将使用已建立的RQC应激反应和非通路翻译事件（如氨基酸错误结合或核糖体移框）的检测方法来探索核糖体在“空核糖体受体位点”情况下的反应。目的是定义新的核糖体应激反应途径，介导细胞对失败tRNA递送的反应。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向核糖体的传递（并避免应激），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和理解这些过程受到损害的人类神经发育疾病。例如，生物技术利用基因表达来生产疫苗、药品和化学原料，但由此对宿主生物造成的生理挑战是相当大的，因为蛋白质合成是细胞中最需要能量的过程之一，产量往往相应地受到影响。为了优化重组蛋白的表达，我们必须了解在分子水平上对翻译需求的管理。该博士生将使用先进的分子生物学方法，包括对酵母（阿伯丁实验室）和哺乳动物细胞（爱丁堡实验室）中的eEF1-tRNA传递系统及其相关的eef1 - β和γ GTP回收装置进行基因组编辑，以了解eEF延伸因子家族的活性如何发挥作用，以优化生物技术、健康和疾病中的蛋白质合成。该研究项目还将结合合成生物学和系统生物学建模，以了解trna是如何传递到核糖体的，以及当外源蛋白质的生物技术表达对系统产生过度需求时所引发的应激反应。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空缺核糖体受体位点的翻译应激，并通过降低蛋白质合成速率来实现蛋白质合成的自体控制，从而实时减少延迟。将提供分子生物学、系统生物学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.