The genetic code is considered one of the most conserved, even immutable, features of life. However, numerous deviations from its canonical form were discovered across the three domains of life over the last 40 years, highlighting the unexpected flexibility of this central process of life. These discoveries and previous work from our laboratory on the evolution of the genetic code inspired this PhD thesis. We explored the hypothesis that synthetic codon ambiguities combined with experimental evolution could lead to the reassignment of rare codons in the yeast S. cerevisiae. For this, we replaced the leucine-CUC decoding tRNA with a mutant serine tRNA, producing yeast strains that incorporate up to 50% of Ser at thousands of Leu-CUC sites, across the entire yeast proteome. Despite the associated high fitness cost, recombinant cells were highly tolerant and capable of adapting to high level Ser incorporation at CUC codons in a long-term evolutionary experiment. We found that adaptation to high Leu-to-Ser mistranslation triggered significant genomic alterations. Indeed, ploidy shifts, increased occurrence of copy number variation (CNVs) events, and aneuploidies were common in the recombinant yeast strains. Transcriptome and translatome analyses identified upregulated ribosomal proteins (RPs) as an unexpected player in the adaptation of yeast to Leu-to-Ser mistranslation at CUC sites. Contrary to expectation, the overexpression of RPs did not increase the protein synthesis rate, and it remains unclear how they contribute to CUC reprogramming or stress tolerance. By achieving 50% of Ser incorporation at Leu-CUC sites on a proteome wide scale, this project pushed the limit of sense codon ambiguity to unprecedented levels, showcasing both its plasticity and the impressive tolerance of yeast to translational errors. It also demonstrates the wide-reaching potential of this approach in synthetic biology and provides new clues about the biology of protein synthesis errors. Moreover, by expanding our understanding of the long-term effects of protein misfolding and aggregation, this work also contributes to the biological basis surrounding the pathophysiology of diseases triggered by these protein alterations.

Genetic code alterations discovered over the last 40 years in bacteria and eukaryotes invalidate the hypothesis that the code is universal and frozen. Mitochondria of various yeast species translate the UGA stop codon as tryptophan (Trp) and leucine (Leu) CUN codons (N = any nucleotide) as threonine (Thr) and fungal CTG clade species reassigned Leu CUG codons to serine and translate them ambiguously in their cytoplasms. This unique sense-to-sense genetic code alteration is mediated by a Ser-tRNA containing a Leu 5'-CAG-3'anticodon (ser-tRNA(CAG)), which is recognized and charged with Ser (~97%) by the seryl-tRNA synthetase (SerRS) and with Leu (~3%) by the leucyl-tRNA synthetase (LeuRS). This unusual tRNA appeared 272 ± 25 million years ago and had a profound impact on the evolution of the CTG clade species. Here, we review the most recent results and concepts arising from the study of this codon reassignment and we highlight how its study is changing our views of the evolution of the genetic code.

The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution.

During the last 30 years, a number of alterations to the standard genetic code have been uncovered both in prokaryotes and eukaryotic nuclear and mitochondrial genomes. But, the study of the evolutionary pathways and molecular mechanisms of codon identity redefinition has been largely ignored due to the assumption that non-standard genetic codes can only evolve through neutral evolutionary mechanisms and that they have no functional significance. The recent discovery of a genetic code change in the genus Candida that evolved through an ambiguous messenger RNA decoding mechanism is bringing that naive assumption to an abrupt end by showing, in a rather dramatic way, that genetic code changes have profound physiological and evolutionary consequences for the species that redefine codon identity. In this paper, the recent data on the evolution of the Candida genetic code are reviewed and an experimental framework based on forced evolution, molecular genetics and comparative and functional genomics methodologies is put forward for the study of non-standard genetic codes and genetic code ambiguity in general. Additionally, the importance of using Saccharomyces cerevisiae as a model organism for elucidating the evolutionary pathway of the Candida and other genetic code changes is emphasised.

The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.

Currently, the teacher is faced with several alternative educational methodologies that make the traditional expository method increasingly improved. Added to this, education has gone through delicate moments at the expense of the new Coronavirus pandemic (SARS-CoV-2). The use of educational games has been highly appreciated for facilitating and motivating student learning. In higher education, the genetic code theme is generally seen as complex and difficult to assimilate due to the vast amount of terms and concepts that need to be assimilated. The objective of this work is to propose a game that facilitates the understanding of the genetic code theme in the genetics discipline. In addition, the game allows you to approach topics of Biochemistry, regarding the characteristics of the structural constitution of amino acids, and other concepts such as essential, non-essential or conditionally essential amino acids. We believe that the use of this activity can captivate students and promote improvement in the teaching and learning process on the subject.