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

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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.