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.     

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.     

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 bias covaries with expression breadth and the rate of synonymous evolution in humans, but this is not evidence for selection.

In numerous species, from bacteria to Drosophila, evidence suggests that selection acts even on synonymous codon usage: codon bias is greater in more abundantly expressed genes, the rate of synonymous evolution is lower in genes with greater codon bias, and there is consistency between genes in the same species in which codons are preferred. In contrast, in mammals, while nonequal use of alternative codons is observed, the bias is attributed to the background variance in nucleotide concentrations, reflected in the similar nucleotide composition of flanking noncoding and exonic third sites. However, a systematic examination of the covariants of codon usage controlling for background nucleotide content has yet to be performed. Here we present a new method to measure codon bias that corrects for background nucleotide content and apply this to 2396 human genes. Nearly all (99%) exhibit a higher amount of codon bias than expected by chance. The patterns associated with selectively driven codon bias are weakly recovered: Broadly expressed genes have a higher level of bias than do tissue-specific genes, the bias is higher for genes with lower rates of synonymous substitutions, and certain codons are repeatedly preferred. However, while these patterns are suggestive, the first two patterns appear to be methodological artifacts. The last pattern reflects in part biases in usage of nucleotide pairs. We conclude that we find no evidence for selection on codon usage in humans.     

Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases.

Codon usage data for 56 Bacillus subtilis genes show that synonymous codon usage in B. subtilis is less biased than in Escherichia coli, or in Saccharomyces cerevisiae. Nevertheless, certain genes with a high codon bias can be identified by correspondence analysis, and also by various indices of codon bias. These genes are very highly expressed, and a general trend (a decrease) in codon bias across genes seems to correspond to decreasing expression level. This, then, may be a general phenomenon in unicellular organisms. The unusually small effect of translational selection on the pattern of codon usage in lowly expressed genes in B. subtilis yields similar dinucleotide frequencies among different codon positions, and on complementary strands. These patterns could arise through selection on DNA structure, but more probably are largely determined by mutation. This prevalence of mutational bias could lead to difficulties in assessing whether open reading frames encode proteins.     

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.     

Bioinformatic analysis of the link between gene composition and expressivity in Saccharomyces cerevisiae and Schizosaccharomyces pombe

The compositional non-randomness was studied in genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both species, codon usage is well correlated with expressivity (measured as the codon adaptation index). Both species generally display higher nucleotide non-randomness in the group of highly expressed genes than in the lowly expressed genes. The highly expressed genes in both species are furthermore characterized by marked peaks in non-randomness at N=3 upstream of start codons, N=2 downstream of start codons and at N=1 and N=7 downstream of stop codons, indicating that these nucleotides may be key elements in translational regulation. Intragenic variation in codon usage was also observed to be linked to expressivity. It is suggested that the firm link between expressivity and codon usage calls for codon optimization. Based on bioinformatic calculations, examples of proteins are given for which codon optimizations might be relevant.     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999     

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.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

Selection Conflicts,Gene Expression,and Codon Usage Trends in Yeast

Synonymous codon usage in yeast appears to be influenced by natural selection on gene expression, as well as regional variation in compositional bias. Because of the large number of potential targets of selection (i.e., most of the codons in the genome) and presumed small selection coefficients, codon usage is an excellent model for studying factors that limit the effectiveness of selection. We use factor analysis to identify major trends in codon usage for 5836 genes in Saccharomyces cerevisiae. The primary factor is strongly correlated with gene expression, consistent with the model that a subset of codons allows for more efficient translation. The secondary factor is very strongly correlated with third codon position GC content and probably reflects regional variation in compositional bias. We find that preferred codon usage decreases in the face of three potential limitations on the effectiveness of selection: reduced recombination rate, increased gene length, and reduced intergenic spacing. All three patterns are consistent with the Hill–Robertson effect (reduced effectiveness of selection among linked targets). A reduction in gene expression in closely spaced genes may also reflect selection conflicts due to antagonistic pleiotropy.     

Unusual codon usage bias in low expression genes of Vibrio cholerae

Positive correlation between gene expression and synonymous codon usage bias is well documented in the literature. However, in the present study of Vibrio cholerae genome, we have identified a group of genes having unusually high codon usage bias despite being low potential expressivity. Our results suggest that codon usage in lowly expressed genes might also be selected on to preferably use non-optimal codons to maintain a low cellular concentration of the proteins that they encode. This would predict that lowly expressed genes are also biased in codon usage, but in a way that is opposite to the bias of highly expressed genes.     

Comparative analyses of codon and amino acid usage in symbiotic island and core genome in nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum

Genes involved in the symbiotic interactions between the nitrogen-fixing endosymbiont Bradyrhizobium japonicum, and its leguminous host are mostly clustered in a symbiotic island (SI), acquired by the bacterium through a process of horizontal transfer. A comparative analysis of the codon and amino acid usage in core and SI genes/proteins of B. japonicum has been carried out in the present study. The mutational bias, translational selection, and gene length are found to be the major sources of variation in synonymous codon usage in the core genome as well as in SI, the strength of translational selection being higher in core genes than in SI. In core proteins, hydrophobicity is the main source of variation in amino acid usage, expressivity and aromaticity being the second and third important sources. But in SI proteins, aromaticity is the chief source of variation, followed by expressivity and hydrophobicity. In SI proteins, both the mean molecular weight and mean aromaticity of individual proteins exhibit significant positive correlation with gene expressivity, which violate the cost-minimization hypothesis. Investigation of nucleotide substitution patterns in B. japonicum and Mesorhizobium loti orthologous genes reveals that both synonymous and non-synonymous sites of highly expressed genes are more conserved than their lowly expressed counterparts and this conservation is more pronounced in the genes present in core genome than in SI.     

Variable strength of translational selection among 12 Drosophila species

Codon usage bias in Drosophila melanogaster genes has been attributed to negative selection of those codons whose cellular tRNA abundance restricts rates of mRNA translation. Previous studies, which involved limited numbers of genes, can now be compared against analyses of the entire gene complements of 12 Drosophila species whose genome sequences have become available. Using large numbers (6138) of orthologs represented in all 12 species, we establish that the codon preferences of more closely related species are better correlated. Differences between codon usage biases are attributed, in part, to changes in mutational biases. These biases are apparent from the strong correlation (r = 0.92, P < 0.001) among these genomes' intronic G + C contents and exonic G + C contents at degenerate third codon positions. To perform a cross-species comparison of selection on codon usage, while accounting for changes in mutational biases, we calibrated each genome in turn using the codon usage bias indices of highly expressed ribosomal protein genes. The strength of translational selection was predicted to have varied between species largely according to their phylogeny, with the D. melanogaster group species exhibiting the strongest degree of selection.     

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.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.