A Comparative Genomics Analysis of Codon Reassignments Reveals a Link with Mitochondrial Proteome Size and a Mechanism of Genetic Code Change Via Suppressor tRNAs

Using a comparative genomics approach we demonstrate a negative correlation between the number of codon reassignments undergone by 222 mitochondrial genomes and the mitochondrial genome size, the number of mitochondrial ORFs, and the sizes of the large and small subunit mitochondrial rRNAs. In addition, we show that the TGA-to-tryptophan codon reassignment, which has occurred 11 times in mitochondrial genomes, is found in mitochondrial genomes smaller than those which have not undergone the reassignment. We therefore propose that mitochondrial codon reassignments occur in a wide range of phyla, particularly in Metazoa, due to a reduced “proteomic constraint” on the mitochondrial genetic code, compared to the nuclear genetic code. The reduced proteomic constraint reflects the small size of the mitochondrial-encoded proteome and allows codon reassignments to occur with less likelihood of lethality. In addition, we demonstrate a striking link between nonsense codon reassignments and the decoding properties of naturally occurring nonsense suppressor tRNAs. This suggests that natural preexisting nonsense suppression facilitated nonsense codon reassignments and constitutes a novel mechanism of genetic code change. These findings explain for the first time the identity of the stop codons and amino acids reassigned in mitochondrial and nuclear genomes. Nonsense suppressor tRNAs provided the raw material for nonsense codon reassignments, implying that the properties of the tRNA anticodon have dictated the identity of nonsense codon reassignments. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Laura Landweber]     

The Mechanisms of Codon Reassignments in Mitochondrial Genetic Codes

Many cases of nonstandard 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 (CD), 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. CD 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 CD. 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 the 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.     

Parallel evolution of the genetic code in arthropod mitochondrial genomes

The genetic code provides the translation table necessary to transform the information contained in DNA into the language of proteins. In this table, a correspondence between each codon and each amino acid is established: tRNA is the main adaptor that links the two. Although the genetic code is nearly universal, several variants of this code have been described in a wide range of nuclear and organellar systems, especially in metazoan mitochondria. These variants are generally found by searching for conserved positions that consistently code for a specific alternative amino acid in a new species. We have devised an accurate computational method to automate these comparisons, and have tested it with 626 metazoan mitochondrial genomes. Our results indicate that several arthropods have a new genetic code and translate the codon AGG as lysine instead of serine (as in the invertebrate mitochondrial genetic code) or arginine (as in the standard genetic code). We have investigated the evolution of the genetic code in the arthropods and found several events of parallel evolution in which the AGG codon was reassigned between serine and lysine. Our analyses also revealed correlated evolution between the arthropod genetic codes and the tRNA-Lys/-Ser, which show specific point mutations at the anticodons. These rather simple mutations, together with a low usage of the AGG codon, might explain the recurrence of the AGG reassignments.     

Codon swapping as a possible evolutionary mechanism

Summary It is apparent in the genetic code that amino acids of similar chemical nature have similar codons. I show how through successive codon captures (multiple rounds of Osawa-Jukes type reassignments), complete codon swappings in an unfavorable genetic code are evolutionarily feasible. This mechanisms could have complemented the ambiguity reduction and the vocabulary extension processes of codon-amino acid assignments. Evolution of wobble rules is implied. Transfer RNA molecules and synthetases may still carry memories of it.     

Searching of Code Space for an Error-Minimized Genetic Code Via Codon Capture Leads to Failure, or Requires At Least 20 Improving Codon Reassignments Via the Ambiguous Intermediate Mechanism

The standard genetic code (SGC) has a fundamental error-minimizing property which has been widely attributed to the action of selection. However, a clear mechanism for how selection can give rise to error minimization (EM) is lacking. A search through a space of alternate codes (code space) via codon reassignments would be required, to select a code optimized for EM. There are two commonly discussed mechanisms of codon reassignment; the Codon Capture mechanism, which proposes a loss of the codon during reassignment, and the Ambiguous Intermediate mechanism, which proposes that the codon underwent an ambiguous phase during reassignment. When searching of code space via the Codon Capture mechanism is simulated, an optimized genetic code can rarely be achieved (0–3.2% of the time) with most searches ending in failure. When code space is searched via the Ambiguous Intermediate mechanism, under constraints derived from empirical observations of codon reassignments from extant genomes, the searches also often end in failure. When a local minimum is avoided and optimization is achieved, 20–41 sequential improving codon reassignments are required. Furthermore, the structures of the optimized codes produced by these simulations differ from the structure of the SGC. These data are challenges for the Adaptive Code hypothesis to address, which proposes that the EM property was directly selected for, and suggests that EM is simply a byproduct of the addition of amino acids to the expanding code, as described by the alternative ‘Emergence’ hypothesis.     

Development of the genetic code: Insights from a fungal codon reassignment

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.     

Codon usage patterns in cytochrome oxidase I across multiple insect orders

Synonymous codon usage bias is determined by a combination of mutational biases, selection at the level of translation, and genetic drift. In a study of mtDNA in insects, we analyzed patterns of codon usage across a phylogeny of 88 insect species spanning 12 orders. We employed a likelihood-based method for estimating levels of codon bias and determining major codon preference that removes the possible effects of genome nucleotide composition bias. Three questions are addressed: (1) How variable are codon bias levels across the phylogeny? (2) How variable are major codon preferences? and (3) Are there phylogenetic constraints on codon bias or preference? There is high variation in the level of codon bias values among the 88 taxa, but few readily apparent phylogenetic patterns. Bias level shifts within the lepidopteran genus Papilio are most likely a result of population size effects. Shifts in major codon preference occur across the tree in all of the amino acids in which there was bias of some level. The vast majority of changes involves double-preference models, however, and shifts between single preferred codons within orders occur only 11 times. These shifts among codons in double-preference models are phylogenetically conservative.     

The comparative method rules! Codon volatility cannot detect positive Darwinian selection using a single genome sequence

All established methods for detecting positive selection at the molecular level rely on comparisons between nucleotide sequences. An exceptional method that purports to detect selection on the basis of a single genomic sequence has recently been proposed. This method uses a measure called "codon volatility," defined for each codon as the ratio between the number of nonsynonymous codons that differ from the codon under study at a single nucleotide position and the number of sense codons that differ from the codon under study at a single nucleotide position. Here, we examine various properties of codon volatility and its derivatives and use simulation of evolutionary processes to determine whether they can be used to detect selective pressures. Codons for only four amino acids (glycine, leucine, arginine, and serine) show any variation in codon volatility. Thus, codon volatility is mainly a proxy for amino acid usage, rather than for codon usage, with 65% of all synonymous changes and 27% of all nonsynonymous changes being undetectable by this measure. Genes identified by the volatility method as being subject to positive selection tend to have idiosyncratic amino acid compositions (e.g., they are glycine rich or arginine poor). An additional property of codon volatility is the near zero variance of its mean expectation, which translates into overestimated statistical significance estimates, especially in the absence of corrections for multiple comparisons. A comparison with measures of selection inferred through comparative methodology reveals no relationship between the results of the two methods. Finally, we show that codon volatility can increase in the absence of positive Darwinian selection; that is, increased codon volatility is not indicative of positive selection.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

Early fixation of an optimal genetic code

The evolutionary forces that produced the canonical genetic code before the last universal ancestor remain obscure. One hypothesis is that the arrangement of amino acid/codon assignments results from selection to minimize the effects of errors (e.g., mistranslation and mutation) on resulting proteins. If amino acid similarity is measured as polarity, the canonical code does indeed outperform most theoretical alternatives. However, this finding does not hold for other amino acid properties, ignores plausible restrictions on possible code structure, and does not address the naturally occurring nonstandard genetic codes. Finally, other analyses have shown that significantly better code structures are possible. Here, we show that if theoretically possible code structures are limited to reflect plausible biological constraints, and amino acid similarity is quantified using empirical data of substitution frequencies, the canonical code is at or very close to a global optimum for error minimization across plausible parameter space. This result is robust to variation in the methods and assumptions of the analysis. Although significantly better codes do exist under some assumptions, they are extremely rare and thus consistent with reports of an adaptive code: previous analyses which suggest otherwise derive from a misleading metric. However, all extant, naturally occurring, secondarily derived, nonstandard genetic codes do appear less adaptive. The arrangement of amino acid assignments to the codons of the standard genetic code appears to be a direct product of natural selection for a system that minimizes the phenotypic impact of genetic error. Potential criticisms of previous analyses appear to be without substance. That known variants of the standard genetic code appear less adaptive suggests that different evolutionary factors predominated before and after fixation of the canonical code. While the evidence for an adaptive code is clear, the process by which the code achieved this optimization requires further attention.     

The role of alternative genetic codes in viral evolution and emergence

Although the ‘universal’ genetic code is widespread among life-forms, a number of diverse lineages have evolved unique codon reassignments. The proteomes of these organisms and organelles must, by necessity, use the same codon assignments. Likewise, for an exogenous genetic element, such as an infecting viral genome, to be accurately and completely expressed with the host's translation system, it must employ the same genetic code. This raises a number of intriguing questions regarding the origin and evolution of viruses. In particular, it is extremely unlikely that viruses of hosts utilizing the universal genetic code would emerge, via cross-species transmission, in hosts utilizing alternative codes, and vice versa. Consequently, more parsimonious scenarios for the origins of such viruses include the prolonged co-evolution of viruses with cellular life, or the escape of genetic material from host genomes. Further, we raise the possibility that emerging viruses provide the selection pressure favoring the use of alternative codes in potential hosts, such that the evolution of a variant genetic code acts as a unique and powerful antiviral strategy. As such, in the face of new emerging viruses, hosts with codon reassignments would have a significant selective advantage compared to hosts utilizing the universal code.     

How Mitochondria Redefine the Code

Annotated, complete DNA sequences are available for 213 mitochondrial genomes from 132 species. These provide an extensive sample of evolutionary adjustment of codon usage and meaning spanning the history of this organelle. Because most known coding changes are mitochondrial, such data bear on the general mechanism of codon reassignment. Coding changes have been attributed variously to loss of codons due to changes in directional mutation affecting the genome GC content (Osawa and Jukes 1988), to pressure to reduce the number of mitochondrial tRNAs to minimize the genome size (Anderson and Kurland 1991), and to the existence of transitional coding mechanisms in which translation is ambiguous (Schultz and Yarus 1994a). We find that a succession of such steps explains existing reassignments well. In particular, (1) Genomic variation in the prevalence of a codon's third-position nucleotide predicts relative mitochondrial codon usage well, though GC content does not. This is because A and T, and G and C, are uncorrelated in mitochondrial genomes. (2) Codons predicted to reach zero usage (disappear) do so more often than expected by chance, and codons that do disappear are disproportionately likely to be reassigned. However, codons predicted to disappear are not significantly more likely to be reassigned. Therefore, low codon frequencies can be related to codon reassignment, but appear to be neither necessary nor sufficient for reassignment. (3) Changes in the genetic code are not more likely to accompany smaller numbers of tRNA genes and are not more frequent in smaller genomes. Thus, mitochondrial codons are not reassigned during demonstrable selection for decreased genome size. Instead, the data suggest that both codon disappearance and codon reassignment depend on at least one other event. This mitochondrial event (leading to reassignment) occurs more frequently when a codon has disappeared, and produces only a small subset of possible reassignments. We suggest that coding ambiguity, the extension of a tRNA's decoding capacity beyond its original set of codons, is the second event. Ambiguity can act alone but often acts in concert with codon disappearance, which promotes codon reassignment. Received: 26 October 2000 / Accepted: 19 January 2001     

Does the Genetic Code Have A Eukaryotic Origin?

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core "house-keeping" functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables-GC and purine contents-of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern-the symmetric pattern-where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes.     

Factors influencing synonymous codon and amino acid usage biases in Mimivirus

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.     

On the Evolution of Redundancy in Genetic Codes

We simulate a deterministic population genetic model for the coevolution of genetic codes and protein-coding genes. We use very simple assumptions about translation, mutation, and protein fitness to calculate mutation-selection equilibria of codon frequencies and fitness in a large asexual population with a given genetic code. We then compute the fitnesses of altered genetic codes that compete to invade the population by translating its genes with higher fitness. Codes and genes coevolve in a succession of stages, alternating between genetic equilibration and code invasion, from an initial wholly ambiguous coding state to a diversified frozen coding state. Our simulations almost always resulted in partially redundant frozen genetic codes. Also, the range of simulated physicochemical properties among encoded amino acids in frozen codes was always less than maximal. These results did not require the assumption of historical constraints on the number and type of amino acids available to codes nor on the complexity of proteins, stereochemical constraints on the translational apparatus, nor mechanistic constraints on genetic code change. Both the extent and timing of amino-acid diversification in genetic codes were strongly affected by the message mutation rate and strength of missense selection. Our results suggest that various omnipresent phenomena that distribute codons over sites with different selective requirements—such as the persistence of nonsynonymous mutations at equilibrium, the positive selection of the same codon in different types of sites, and translational ambiguity—predispose the evolution of redundancy and of reduced amino acid diversity in genetic codes. Received: 21 December 2000 / Accepted: 12 March 2001     

Comparative Genomics of Trypanosomatid Pathogens using Codon Usage Bias

It is well known that an amino acid can be encoded by more than one codon, called synonymous codons. The preferential use of one particular codon for coding an amino acid is referred to as codon usage bias (CUB). A quantitative analytical method, CUB and a related tool, Codon Adaptative Index have been applied to comparatively study whole genomes of a few pathogenic Trypanosomatid species. This quantitative attempt is of direct help in the comparison of qualitative features like mutational and translational selection. Pathogens of the Leishmania and Trypanosoma genus cause debilitating disease and suffering in human beings and animals. Of these, whole genome sequences are available for only five species. The complete coding sequences (CDS), highly expressed, essential and low expressed genes have all been studied for their CUB signature. The codon usage bias of essential genes and highly expressed genes show distribution similar to codon usage bias of all CDSs in Trypanosomatids. Translational selection is the dominant force selecting the preferred codon, and selection due to mutation is negligible. In contrast to an earlier study done on these pathogens, it is found in this work that CUB and CAI may be used to distinguish the Trypanosomatid genomes at the sub-genus level. Further, CUB may effectively be used as a signature of the species differentiation by using Principal Component Analysis (PCA).

Abbreviations

CUB - Codon Usage Bias, CAI - Codon Adaptative Index, CDS - Coding sequences, t-RNA - Transfer RNA, PCA - Principal Component Analysis.     

Strong Heterogeneity in Nucleotidic Composition and Codon Bias in the Pea Aphid (<Emphasis Type="Italic">Acyrthosiphon pisum)</Emphasis> Shown by EST-Based Coding Genome Reconstruction

The aim of this study was to analyze patterns of nucleotidic composition and codon usage in the pea aphid genome (Acyrthosiphon pisum). A collection of 60,000 expressed sequence tags (ESTs) in the pea aphid has been used to automatically reconstruct 5809 coding sequences (CDSs), based on similarity with known proteins and on coding style recognition. Reconstructions were manually checked for ribosomal proteins, leading to tentatively reconstruct the nea-complete set of this category. Pea aphid coding sequences showed a shift toward AT (especially at the third codon position) compared to drosophila homologues. Genes with a putative high level of expression (ribosomal and other genes with high EST support) remained more GC3-rich and had a distinct codon usage from bulk sequences: they exhibited a preference for C-ending codons and CGT (for arginine), which thus appeared optimal for translation. However, the discrimination was not as strong as in drosophila, suggesting a reduced degree of translational selection. The space of variation in codon usage for A. pisum appeared to be larger than in drosophila, with a substantial fraction of genes that remained GC3-rich. Some of those (in particular some structural proteins) also showed high levels of codon bias and a very strong preference for C-ending codons, which could be explained either by strong translational selection or by other mechanisms. Finally, genomic traces were analyzed to build 206 fragments containing a full CDS, which allowed studying the correlations between GC contents of coding and those of noncoding (flanking and introns) sequences.     

Relationship between amino acid changes in mitochondrial ATP6 and life-history variation in anguillid eels

Mitochondrial genes are part of the oxidative phosphorylation pathway and important for energy production. Although evidence for positive selection at the mitochondrial level exists, few studies have investigated the link between amino acid changes and phenotype. Here we test the hypothesis that differences in two life-history related traits, migratory distance between spawning and foraging areas and larval phase duration, are associated with divergent selection within the mitochondrial ATP6 gene in anguillid eels. We compare amino acid changes among 18 species with the sequence of the putative ancestral species, believed to have shown short migratory distance and larval phase duration. We find positive correlations between both life-history related traits and (i) the number of amino acid changes and (ii) the strength of the combined physico-chemical and structural changes at positions previously identified as candidates for positive selection. This supports a link between genotype and phenotype driven by positive selection at ATP6.