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.

我们的研究项目侧重于真核细胞中基因表达的机制。尽管被认为不如蛋白质修饰那么频繁，但细胞利用错误翻译在翻译水平上产生蛋白质组多样性。当蛋白质合成过程中掺入与标准遗传密码指定的氨基酸不同的氨基酸时，就会发生误译。误译的频率随着不同的环境条件而增加，许多生物体将误译作为一种适应性反应。 tRNA 变异也会导致误译增加。由于 tRNA 是携带氨基酸并解码遗传信息的中间分子，其序列的改变可能会导致它们携带错误的氨基酸或误解基因序列。误译率可达 8-10%，可导致蛋白质展开并破坏蛋白质的细胞补体。细胞能够容忍错误翻译的 tRNA 变体，因为蛋白质质量控​​制途径可以消除或隔离有毒的错误制造蛋白质。此外，几乎所有生物体中都富含 tRNA 编码基因。这些“冗余”基因提供了容忍误译tRNA的缓冲液，也为误译tRNA的进化提供了原材料。我们研究计划的总体假设是，误译会增加遗传多样性并影响生物体的生存和对环境的反应。此外，错误翻译的 tRNA 因其在合成生物学和生物技术应用中的实用性而具有重要意义，因为它们通过产生“统计蛋白质”来扩展蛋白质多样性。尽管统计蛋白质是从相同的遗传信息翻译而来，但它们是分子的异质混合物，其氨基酸的比例差异很小。因此，它们可以表现出比均质形式更广泛的活性和底物特异性。此外，诱导翻译错误被用来研究氨基酸取代对体内细胞功能的影响。对错误翻译的研究还揭示了遗传密码进化、翻译过程以及细胞如何应对蛋白质稳态丧失的重要方面。我们专注于酿酒酵母中 tRNA 的错误翻译，这是研究机制的理想模型，也是合成生物学和生物技术领域有吸引力的生物体。我们的研究计划旨在开发工具来利用误译和统计蛋白质在研究和加拿大生物技术行业中的使用，并了解误译介导的基因表达变化如何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，确定导致错误翻译 tRNA 毒性的因素，识别增加对错误翻译的耐受性的基因，并设计一个用于调节错误翻译 tRNA 表达的系统。