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.

我们的研究项目重点是真核细胞中基因表达的机制。虽然认为不如蛋白质修饰频繁，但细胞使用误译在翻译水平上产生蛋白质组多样性。当在蛋白质合成过程中掺入了与标准遗传密码所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变体也有助于增加误译。由于tRNA是携带氨基酸和解码遗传信息的中间分子，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。误译，可以接近8- 10%，可以导致蛋白质解折叠并破坏蛋白质的细胞补体。细胞容忍错误翻译的tRNA变体，因为蛋白质质量控制途径消除或隔离有毒的错误蛋白质。此外，tRNA编码基因在几乎所有生物中都很丰富。这些“冗余”基因提供了一个缓冲区，以容忍错误翻译的tRNA，也提供了原材料的演变错误翻译的tRNA。我们研究项目的首要假设是，误译增加了遗传多样性，影响了生物体的生存和对环境的反应。此外，错误翻译的tRNA是重要的，因为它们在合成生物学和生物技术应用中的实用性，因为它们通过创建“统计蛋白质”来扩大蛋白质多样性。虽然从相同的遗传信息翻译，统计蛋白质是异质混合物的分子不同的氨基酸的一小部分。因此，它们可以显示比同质形式更广泛的活性和底物特异性。此外，诱导翻译错误被用于研究氨基酸取代对体内细胞功能的影响。对误译的研究还揭示了遗传密码进化的重要方面，翻译过程以及细胞如何科普蛋白质稳态的丧失。我们主要研究酵母S.酿酒酵母是研究机制的理想模型，也是合成生物学和生物技术的一种有吸引力的生物。我们的研究计划旨在开发工具，利用在研究和加拿大生物技术行业中使用误译和统计蛋白质，并了解通过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，确定导致错误翻译的tRNA毒性的因素，鉴定增加对错误翻译的耐受性的基因，并设计一个系统来调节错误翻译的tRNA的表达。