Mistranslation and Synthetic Biology

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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.