Transfer RNA Gene Numbers may not be Completely Responsible for the Codon Usage Bias in Asparagine, Isoleucine, Phenylalanine, and Tyrosine in the High Expression Genes in Bacteria

It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β′, respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.     

Intragenomic variation in non-adaptive nucleotide biases causes underestimation of selection on synonymous codon usage

Patterns of non-uniform usage of synonymous codons vary across genes in an organism and between species across all domains of life. This codon usage bias (CUB) is due to a combination of non-adaptive (e.g. mutation biases) and adaptive (e.g. natural selection for translation efficiency/accuracy) evolutionary forces. Most models quantify the effects of mutation bias and selection on CUB assuming uniform mutational and other non-adaptive forces across the genome. However, non-adaptive nucleotide biases can vary within a genome due to processes such as biased gene conversion (BGC), potentially obfuscating signals of selection on codon usage. Moreover, genome-wide estimates of non-adaptive nucleotide biases are lacking for non-model organisms. We combine an unsupervised learning method with a population genetics model of synonymous coding sequence evolution to assess the impact of intragenomic variation in non-adaptive nucleotide bias on quantification of natural selection on synonymous codon usage across 49 Saccharomycotina yeasts. We find that in the absence of a priori information, unsupervised learning can be used to identify genes evolving under different non-adaptive nucleotide biases. We find that the impact of intragenomic variation in non-adaptive nucleotide bias varies widely, even among closely-related species. We show that the overall strength and direction of translational selection can be underestimated by failing to account for intragenomic variation in non-adaptive nucleotide biases. Interestingly, genes falling into clusters identified by machine learning are also physically clustered across chromosomes. Our results indicate the need for more nuanced models of sequence evolution that systematically incorporate the effects of variable non-adaptive nucleotide biases on codon frequencies.     

Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonymous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa, and L. lactis as their primary host. We use the concept of a “genome landscape,” which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such as GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.     

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.     

Signatures of optimal codon usage in metabolic genes inform budding yeast ecology

Reverse ecology is the inference of ecological information from patterns of genomic variation. One rich, heretofore underutilized, source of ecologically relevant genomic information is codon optimality or adaptation. Bias toward codons that match the tRNA pool is robustly associated with high gene expression in diverse organisms, suggesting that codon optimization could be used in a reverse ecology framework to identify highly expressed, ecologically relevant genes. To test this hypothesis, we examined the relationship between optimal codon usage in the classic galactose metabolism (GAL) pathway and known ecological niches for 329 species of budding yeasts, a diverse subphylum of fungi. We find that optimal codon usage in the GAL pathway is positively correlated with quantitative growth on galactose, suggesting that GAL codon optimization reflects increased capacity to grow on galactose. Optimal codon usage in the GAL pathway is also positively correlated with human-associated ecological niches in yeasts of the CUG-Ser1 clade and with dairy-associated ecological niches in the family Saccharomycetaceae. For example, optimal codon usage of GAL genes is greater than 85% of all genes in the genome of the major human pathogen Candida albicans (CUG-Ser1 clade) and greater than 75% of genes in the genome of the dairy yeast Kluyveromyces lactis (family Saccharomycetaceae). We further find a correlation between optimization in the GALactose pathway genes and several genes associated with nutrient sensing and metabolism. This work suggests that codon optimization harbors information about the metabolic ecology of microbial eukaryotes. This information may be particularly useful for studying fungal dark matter—species that have yet to be cultured in the lab or have only been identified by genomic material.

Bias toward codons that match the tRNA pool is robustly associated with high gene expression in diverse organisms, suggesting that codon optimization could be used in a reverse ecology framework to identify ecologically relevant genes. This study finds that this is indeed the case for 329 species of budding yeasts, suggesting that codon optimization harbors information about the metabolic ecology of microbial eukaryotes.     

Nature of selective constraints on synonymous codon usage of rice differs in GC-poor and GC-rich genes

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.     

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     

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.     

Universal Pattern and Diverse Strengths of Successive Synonymous Codon Bias in Three Domains of Life, Particularly Among Prokaryotic Genomes

There has been significant progress in understanding the process of protein translation in recent years. One of the best examples is the discovery of usage bias in successive synonymous codons and its role in eukaryotic translation efficiency. We observed here a similar type of bias in the other two life domains, bacteria and archaea, although the bias strength was much smaller than in eukaryotes. Among 136 prokaryotic genomes, 98 were found to have significant bias from random use of successive synonymous codons with Z scores larger than three. Furthermore, significantly different bias strengths were found between prokaryotes grouped by various genomic or biochemical characteristics. Interestingly, the bias strength measured by a general Z score could be fitted well (R = 0.83, P < 10⁻¹⁵) by three genomic variables: genome size, G + C content, and tRNA gene number based on multiple linear regression. A different distribution of synonymous codon pairs between protein-coding genes and intergenic sequences suggests that bias is caused by translation selection. The present results indicate that protein translation is tuned by codon (pair) usage, and the intensity of the regulation is associated with genome size, tRNA gene number, and G + C content.     

Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias

Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do `prefer' optimal codons. Nucleic Acids Res. 17, 5029–5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525–2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H. influenzae and M. tuberculosis are similar to those of E. coli, implying that though the composition of all three codon positions is governed by a selection-mutation balance, the mutational pressure has little influence in the choice of bases at the first two codon positions, even in organisms with highly biased base compositions.     

Variation in the strength of selected codon usage bias among bacteria

Among bacteria, many species have synonymous codon usage patterns that have been influenced by natural selection for those codons that are translated more accurately and/or efficiently. However, in other species selection appears to have been ineffective. Here, we introduce a population genetics-based model for quantifying the extent to which selection has been effective. The approach is applied to 80 phylogenetically diverse bacterial species for which whole genome sequences are available. The strength of selected codon usage bias, S, is found to vary substantially among species; in 30% of the genomes examined, there was no significant evidence that selection had been effective. Values of S are highly positively correlated with both the number of rRNA operons and the number of tRNA genes. These results are consistent with the hypothesis that species exposed to selection for rapid growth have more rRNA operons, more tRNA genes and more strongly selected codon usage bias. For example, Clostridium perfringens, the species with the highest value of S, can have a generation time as short as 7 min.     

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 shapes codon usage in the GC-rich genome of Chlamydomonas reinhardtii

In unicellular species codon usage is determined by mutational biases and natural selection. Among prokaryotes, the influence of these factors is different if the genome is skewed towards AT or GC, since in AT-rich organisms translational selection is absent. On the other hand, in AT-rich unicellular eukaryotes the two factors are present. In order to understand if GC-rich genomes display a similar behavior, the case of Chlamydomonas reinhardtii was studied. Since we found that translational selection strongly influences codon usage in this species, we conclude that there is not a common pattern among unicellular organisms.     

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.     

Universal function-specificity of codon usage

Synonymous codon usage has long been known as a factor that affects average expression level of proteins in fast-growing microorganisms, but neither its role in dynamic changes of expression in response to environmental changes nor selective factors shaping it in the genomes of higher eukaryotes have been fully understood. Here, we propose that codon usage is ubiquitously selected to synchronize the translation efficiency with the dynamic alteration of protein expression in response to environmental and physiological changes. Our analysis reveals that codon usage is universally correlated with gene function, suggesting its potential contribution to synchronized regulation of genes with similar functions. We directly show that coexpressed genes have similar synonymous codon usages within the genomes of human, yeast, Caenorhabditis elegans and Escherichia coli. We also demonstrate that perturbing the codon usage directly affects the level or even direction of changes in protein expression in response to environmental stimuli. Perturbing tRNA composition also has tangible phenotypic effects on the cell. By showing that codon usage is universally function-specific, our results expand, to almost all organisms, the notion that cells may need to dynamically alter their intracellular tRNA composition in order to adapt to their new environment or physiological role.     

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.     

Rapid divergence of codon usage patterns within the rice genome

Background

Synonymous codon usage varies widely between genomes, and also between genes within genomes. Although there is now a large body of data on variations in codon usage, it is still not clear if the observed patterns reflect the effects of positive Darwinian selection acting at the level of translational efficiency or whether these patterns are due simply to the effects of mutational bias. In this study, we have included both intra-genomic and inter-genomic comparisons of codon usage. This allows us to distinguish more efficiently between the effects of nucleotide bias and translational selection.

Results

We show that there is an extreme degree of heterogeneity in codon usage patterns within the rice genome, and that this heterogeneity is highly correlated with differences in nucleotide content (particularly GC content) between the genes. In contrast to the situation observed within the rice genome, Arabidopsis genes show relatively little variation in both codon usage and nucleotide content. By exploiting a combination of intra-genomic and inter-genomic comparisons, we provide evidence that the differences in codon usage among the rice genes reflect a relatively rapid evolutionary increase in the GC content of some rice genes. We also noted that the degree of codon bias was negatively correlated with gene length.

Conclusion

Our results show that mutational bias can cause a dramatic evolutionary divergence in codon usage patterns within a period of approximately two hundred million years.The heterogeneity of codon usage patterns within the rice genome can be explained by a balance between genome-wide mutational biases and negative selection against these biased mutations. The strength of the negative selection is proportional to the length of the coding sequences. Our results indicate that the large variations in synonymous codon usage are not related to selection acting on the translational efficiency of synonymous codons.