Synonymous Codon Usage, Accuracy of Translation, and Gene Length in Caenorhabditis elegans

In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis. However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation. Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species. As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens. Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes. This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases. The relationship between codon bias and gene length remains unexplained. Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage. Received: 18 June 2000 / Accepted: 10 November 2000     

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.     

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.     

Codon usage bias: causative factors,quantification methods and genome‐wide patterns: with emphasis on insect genomes

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole‐genome sequencing of numerous species, both prokaryotes and eukaryotes, genome‐wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole‐genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome‐sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome‐sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance.     

Codon usage in mitochondrial genomes: distinguishing context-dependent mutation from translational selection

We analyze the frequencies of synonymous codons in animal mitochondrial genomes, focusing particularly on mammals and fish. The frequencies of bases at 4-fold degenerate sites are found to be strongly influenced by context-dependent mutation, which causes correlations between pairs of neighboring bases. There is a pattern of excess of certain dinucleotides and deficit of others that is consistent across large numbers of species, despite the wide variation of single-nucleotide frequencies among species. In many bacteria, translational selection is an important influence on codon usage. In order to test whether translational selection also plays a role in mitochondria, we need to control for context-dependent mutation. Selection for translational accuracy can be detected by comparison of codon usage in conserved and variable sites in the same genes. We give a test of this type that works in the presence of context-dependent mutation. There is very little evidence for translational accuracy selection in the mitochondrial genes considered here. Selection for translational efficiency might lead to preference for codons that match the limited repertoire of anticodons on the mitochondrial tRNAs. This is difficult to detect because the effect would usually be in the same direction in comparable to codon families and so would not cause an observable difference in codon usage between families. Several lines of evidence suggest that this type of selection is weak in most cases. However, we found several cases where unusual bases occur at the wobble position of the tRNA, and in these cases, some evidence for selection on codon usage was found. We discuss the way that these unusual cases are associated with codon reassignments in the mitochondrial genetic code.     

Factors influencing the synonymous codon and amino acid usage bias in AT-rich Pseudomonas aeruginosa phage PhiKZ

To reveal how the AT-rich genome of bacteriophage PhiKZ has been shaped in order to carryout its growth in the GC-rich host Pseudomonas aeruginosa,synonymous codon and amino acid usage bias ofPhiKZ was investigated and the data were compared with that of P.aeruginosa.It was found that synonymouscodon and amino acid usage of PhiKZ was distinct from that of P.aeruginosa.In contrast to P.aeruginosa,the third codon position of the synonymous codons of PhiKZ carries mostly A or T base;codon usage biasin PhiKZ is dictated mainly by mutational bias and,to a lesser extent,by translational selection.A clusteranalysis of the relative synonymous codon usage values of 16 myoviruses including PhiKZ shows that PhiKZis evolutionary much closer to Escherickia coli phage T4.Further analysis reveals that the three factors ofmean molecular weight,aromaticity and cysteine content are mostly responsible for the variation of aminoacid usage in PhiKZ proteins,whereas amino acid usage of P.aeruginosa proteins is mainly governed bygrand average of hydropathicity,aromaticity and cysteine content.Based on these observations,we suggestthat codons of the phage-like PhiKZ have evolved to preferentially incorporate the smaller amino acid residuesinto their proteins during translation,thereby economizing the cost of its development in GC-rich P.aeruginosa.     

The Selective Advantage of Synonymous Codon Usage Bias in Salmonella

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10⁻⁴ per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.     

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d_N/d_S values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.     

Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms

Gene expression is known to correlate with degree of codon bias in many unicellular organisms. However, such correlation is absent in some organisms. Recently we demonstrated that inverted complementary repeats within coding DNA sequence must be considered for proper estimation of translation efficiency, since they may form secondary structures that obstruct ribosome movement. We have developed a program for estimation of potential coding DNA sequence expression in defined unicellular organism using its genome sequence. The program computes elongation efficiency index. Computation is based on estimation of coding DNA sequence elongation efficiency, taking into account three key factors: codon bias, average number of inverted complementary repeats, and free energy of potential stem-loop structures formed by the repeats. The influence of these factors on translation is numerically estimated. An optimal proportion of these factors is computed for each organism individually. Quantitative translational characteristics of 384 unicellular organisms (351 bacteria, 28 archaea, 5 eukaryota) have been computed using their annotated genomes from NCBI GenBank. Five potential evolutionary strategies of translational optimization have been determined among studied organisms. A considerable difference of preferred translational strategies between Bacteria and Archaea has been revealed. Significant correlations between elongation efficiency index and gene expression levels have been shown for two organisms (S. cerevisiae and H. pylori) using available microarray data. The proposed method allows to estimate numerically the coding DNA sequence translation efficiency and to optimize nucleotide composition of heterologous genes in unicellular organisms. Availability: http://www.mgs.bionet.nsc.ru/mgs/programs/eei-calculator/.     

Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation

Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.     

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high‐level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.     

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon-anticodon interaction, all consistent with more efficient translation.     

Solving the riddle of codon usage preferences: a test for translational selection

Translational selection is responsible for the unequal usage of synonymous codons in protein coding genes in a wide variety of organisms. It is one of the most subtle and pervasive forces of molecular evolution, yet, establishing the underlying causes for its idiosyncratic behaviour across living kingdoms has proven elusive to researchers over the past 20 years. In this study, a statistical model for measuring translational selection in any given genome is developed, and the test is applied to 126 fully sequenced genomes, ranging from archaea to eukaryotes. It is shown that tRNA gene redundancy and genome size are interacting forces that ultimately determine the action of translational selection, and that an optimal genome size exists for which this kind of selection is maximal. Accordingly, genome size also presents upper and lower boundaries beyond which selection on codon usage is not possible. We propose a model where the coevolution of genome size and tRNA genes explains the observed patterns in translational selection in all living organisms. This model finally unifies our understanding of codon usage across prokaryotes and eukaryotes. Helicobacter pylori, Saccharomyces cerevisiae and Homo sapiens are codon usage paradigms that can be better understood under the proposed model.     

Synonymous Codon Choices in the Extremely GC-Poor Genome of Plasmodium falciparum: Compositional Constraints and Translational Selection

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection. Received: 10 November 1998 / Accepted: 28 January 1999     

Translational selection on codon usage in the genus Aspergillus

Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression.     

Variation in global codon usage bias among prokaryotic organisms is associated with their lifestyles

Background  

It is widely acknowledged that synonymous codons are used unevenly among genes in a genome. In organisms under translational selection, genes encoding highly expressed proteins are enriched with specific codons. This phenomenon, termed codon usage bias, is common to many organisms and has been recognized as influencing cellular fitness. This suggests that the global extent of codon usage bias of an organism might be associated with its phenotypic traits.     

Translational selection on SHH genes

Codon usage bias has been observed in various organisms. In this study, the correlation between SHH genes expression in some tissues and codon usage features was analyzed by bioinformatics. We found that translational selection may act on compositional features of this set of genes.     

Balanced codon usage optimizes eukaryotic translational efficiency

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon-tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology.     

Genomic choice of codons in 16 microbial species

We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya. We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection. Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species. Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, N(C)' (effective number of codons reported to neutral model). Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods. We thus propose set of codons probably preferred by selection for nine other species. In the four last species, no clear preference can be evidenced. Finally, we characterize variation of codon usage over functional categories. We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources.     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.