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The genetic code has been proposed to be a ‘frozen accident,’ but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force that likely helped drive these codons to low frequency and enable their reassignment., eLife digest All life forms rely on a ‘code’ to translate their genetic information into proteins. This code relies on limited permutations of three nucleotides – the building blocks that form DNA and other types of genetic information. Each ‘triplet’ of nucleotides – or codon – encodes a specific amino acid, the basic component of proteins. Reading the sequence of codons in the right order will let the cell know which amino acid to assemble next on a growing protein. For instance, the codon CGG – formed of the nucleotides guanine (G) and cytosine (C) – codes for the amino acid arginine. From bacteria to humans, most life forms rely on the same genetic code. Yet certain organisms have evolved to use slightly different codes, where one or several codons have an altered meaning. To better understand how alternative genetic codes have evolved, Shulgina and Eddy set out to find more organisms featuring these altered codons, creating a new software called Codetta that can analyze the genome of a microorganism and predict the genetic code it uses. Codetta was then used to sift through the genetic information of 250,000 microorganisms. This was made possible by the sequencing, in recent years, of the genomes of hundreds of thousands of bacteria and other microorganisms – including many never studied before. These analyses revealed five groups of bacteria with alternative genetic codes, all of which had changes in the codons that code for arginine. Amongst these, four had genomes with a low proportion of guanine and cytosine nucleotides. This may have made some guanine and cytosine-rich arginine codons very rare in these organisms and, therefore, easier to be reassigned to encode another amino acid. The work by Shulgina and Eddy demonstrates that Codetta is a new, useful tool that scientists can use to understand how genetic codes evolve. In addition, it can also help to ensure the accuracy of widely used protein databases, which assume which genetic code organisms use to predict protein sequences from their genomes.

Genetic code deviations involving stop codons have been previously reported in mitochondrial genomes of several green plants (Viridiplantae), most notably chlorophyte algae (Chlorophyta). However, as changes in codon recognition from one amino acid to another are more difficult to infer, such changes might have gone unnoticed in particular lineages with high evolutionary rates that are otherwise prone to codon reassignments. To gain further insight into the evolution of the mitochondrial genetic code in green plants, we have conducted an in-depth study across mtDNAs from 51 green plants (32 chlorophytes and 19 streptophytes). Besides confirming known stop-to-sense reassignments, our study documents the first cases of sense-to-sense codon reassignments in Chlorophyta mtDNAs. In several Sphaeropleales, we report the decoding of AGG codons (normally arginine) as alanine, by tRNA(CCU) of various origins that carry the recognition signature for alanine tRNA synthetase. In Chromochloris, we identify tRNA variants decoding AGG as methionine and the synonymous codon CGG as leucine. Finally, we find strong evidence supporting the decoding of AUA codons (normally isoleucine) as methionine in Pycnococcus. Our results rely on a recently developed conceptual framework (CoreTracker) that predicts codon reassignments based on the disparity between DNA sequence (codons) and the derived protein sequence. These predictions are then validated by an evaluation of tRNA phylogeny, to identify the evolution of new tRNAs via gene duplication and loss, and structural modifications that lead to the assignment of new tRNA identities and a change in the genetic code.

Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.

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. published

The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of “neutral emergence”. The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these “pseudaptations”, and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an “unfreezing” of the codon – amino acid mapping that defines the genetic code, consistent with Crick’s Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content between organisms, a selective pressure in the evolution of sexual reproduction, and differences in translational fidelity. Lastly, the utility of the concept of an informational constraint to other diverse fields of research is explored.

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research. We thank Alexander Johnson for providing the C. albicans strains and plasmids and Judith Berman, Csaba Pál, and Dieter Söll for their useful comments and suggestions on the manuscript. The study was funded by the European Union Framework Program 7 (EUFP7) Sybaris Consortium Project 242220 and the Portuguese Science Foundation through Fundo Europeu de Desenvolvimento Regional (FEDER/FCT) Project PTDC/BIA-MIC/099826/2008. published

Many cases of non-standard genetic codes are known in mitochondrial genomes. We carry out analysis of phylogeny and codon usage of organisms for which the complete mitochondrial genome is available, and we determine the most likely mechanism for codon reassignment in each case. Reassignment events can be classified according to the gain-loss framework. The gain represents the appearance of a new tRNA for the reassigned codon or the change of an existing tRNA such that it gains the ability to pair with the codon. The loss represents the deletion of a tRNA or the change in a tRNA so that it no longer translates the codon. One possible mechanism is Codon Disappearance, where the codon disappears from the genome prior to the gain and loss events. In the alternative mechanisms the codon does not disappear. In the Unassigned Codon mechanism, the loss occurs first, whereas in the Ambiguous Intermediate mechanism, the gain occurs first. Codon usage analysis gives clear evidence of cases where the codon disappeared at the point of the reassignment and also cases where it did not disappear. Codon disappearance is the probable explanation for stop to sense reassignments and a small number of reassignments of sense codons. However, the majority of sense to sense reassignments cannot be explained by codon disappearance. In the latter cases, by analysis of the presence or absence of tRNAs in the genome and of the changes in tRNA sequences, it is sometimes possible to distinguish between the Unassigned Codon and Ambiguous Intermediate mechanisms. We emphasize that not all reassignments follow the same scenario and that it is necessary to consider the details of each case carefully., 53 pages (45 pages, including 4 figures + 8 pages of supplementary information). To appear in J.Mol.Evol