Mistranslation and Synthetic Biology

误译与合成生物学

• 批准号: RGPIN-2020-07046
• 负责人: Brandl, Christopher
• 金额: $ 2.33万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744067
• 关键词: Mistranslation; Synthetic; Biology

Our research program focuses on mechanisms of gene expression in eukaryotic cells. Though considered less frequently than protein modification, cells generate proteome diversity at the translational level using mistranslation. Mistranslation occurs when an amino acid different from one specified by the standard genetic code is incorporated during protein synthesis. The frequency of mistranslation increases in response to different environmental conditions, with mistranslation being used by many organisms as an adaptive response. tRNA variants also contribute to increased mistranslation. As tRNAs are the intermediate molecules that carry amino acids and decode genetic information, changes to their sequence may cause them to carry the wrong amino acid or misinterpret the gene sequence. Mistranslation, which can approach 8-10%, can lead to protein unfolding and disrupt the cellular complement of proteins. Cells tolerate mistranslating tRNA variants because protein quality control pathways eliminate or sequester toxic mis-made proteins. In addition, tRNA-encoding genes are abundant in nearly all organisms. These "redundant" genes provide a buffer to tolerate mistranslating tRNAs and also provide the raw material for the evolution of mistranslating tRNAs. The overarching hypothesis of our research program is that mistranslation increases genetic diversity and impacts an organism's survival and response to their environment. Furthermore, mistranslating tRNAs are significant because of their utility in synthetic biology and biotechnology applications, since they expand protein diversity by creating "statistical proteins". Although translated from the same genetic message, statistical proteins are heterogenous mixtures with molecules differing by a small proportion of amino acids. As such, they can display a wider range of activities and substrate specificities than the homogeneous form. In addition, inducing translational errors is being used to study the impact of amino acid substitutions on cellular function in vivo. Studies of mistranslation also reveal important aspects of the evolution of the genetic code, the process of translation and how cells cope with loss of protein homeostasis. We focus on mistranslating tRNAs in the yeast S. cerevisiae, an ideal model for studies into mechanism and an attractive organism for synthetic biology and biotechnology. Our research program aims to develop tools to exploit the use of mistranslation and statistical proteins in both research and the Canadian biotechnology industry, and to understand how gene expression changes mediated through mistranslation influence the biology of eukaryotic organisms. To achieve this, we will develop tRNA variants to increase the number of possible amino acid substitutions, determine the factors that contribute to the toxicity of a mistranslating tRNA, identify genes that increase tolerance to mistranslation and engineer a system for regulated expression of mistranslating tRNAs.

我们的研究计划着重于真核细胞中基因表达的机制。尽管被认为比蛋白质修饰的频率少，但细胞在翻译水平上产生蛋白质组的多样性，使用误译。当蛋白质合成过程中纳入标准遗传密码的氨基酸与标准遗传密码指定的氨基酸不同时，就会发生错误翻译。误译的频率随着不同环境条件的响应而增加，许多生物体将误倾式用作适应性反应。 tRNA变体也有助于增加误译。由于TRNA是携带氨基酸并解码遗传信息的中间分子，因此其序列的变化可能导致它们携带错误的氨基酸或误解基因序列。可能接近8-10％的失误可能导致蛋白质展开并破坏蛋白质的细胞补体。细胞耐受误倾式tRNA变体，因为蛋白质质量控​​制途径消除或隔离了有毒的错误制造的蛋白质。另外，在几乎所有生物体中，tRNA编码基因都很丰富。这些“冗余”基因提供了一种缓冲液来耐受误误tRNA的缓冲，并且还为误译TRNA的进化提供了原材料。我们的研究计划的总体假设是，误译可以增加遗传多样性，并影响生物体对环境的生存和反应。此外，由于它们在合成生物学和生物技术应用方面的实用性，因此误导性trnas非常重要，因为它们通过创建“统计蛋白质”来扩展蛋白质多样性。尽管从相同的遗传信息翻译，但统计蛋白是异质混合物，分子与少量氨基酸不同。因此，与均匀形式相比，它们可以显示更广泛的活动和底物特异性。另外，诱导翻译误差被用于研究氨基酸取代对体内细胞功能的影响。误译研究还揭示了遗传密码演变的重要方面，翻译过程以及细胞如何应对蛋白质稳态的丧失。我们专注于在酿酒酵母中误译TRNA，这是研究机制和有吸引力的合成生物学和生物技术的理想模型。我们的研究计划旨在开发工具，以利用研究和加拿大生物技术行业中使用误译和统计蛋白的使用，并了解基因表达如何通过误导来改变介导的基因表达会影响真核生物的生物学。为了实现这一目标，我们将开发tRNA变体以增加可能的氨基酸取代的数量，确定导致误倾式tRNA的毒性的因素，确定基因增加对误倾式的耐受性的基因，并设计一种用于调节误导性TRNA表达表达的系统。