Synonymous but not the same: the causes and consequences of codon bias

Despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. As a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. Although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. Ongoing work to quantify the dynamics of initiation and elongation is as important for understanding natural synonymous variation as it is for designing transgenes in applied contexts.     

Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection

In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.     

Synonymous codon bias is related to gene length in Escherichia coli: selection for translational accuracy?

The levels of synonymous codon bias is shown to be positively correlated to gene length in Escherichia coli genes which are thought to be expressed at similar levels; these are genes whose products are present in multimeric proteins in equimolar amounts. It is argued that the positive correlation could be caused by selection to avoid missense errors during translation. Since the cost of producing a protein is proportional to its length, selection in favor of codons which increase accuracy should be greater in longer genes, and long genes should therefore have higher synonymous codon bias. It is also shown that there is variation in synonymous codon use which is independent of either expression level, gene length, amino acid composition, or chromosomal location. This variation is consistent with selection for translational accuracy but may have other origins.      

Synonymous codon usage is subject to selection in thermophilic bacteria

The patterns of synonymous codon usage, both within and among genomes, have been extensively studied over the past two decades. Despite the accumulating evidence that natural selection can shape codon usage, it has not been possible to link a particular pattern of codon usage to a specific external selective force. Here, we have analyzed the patterns of synonymous codon usage in 40 completely sequenced prokaryotic genomes. By combining the genes from several genomes (more than 80 000 genes in all) into a single dataset for this analysis, we were able to investigate variations in codon usage, both within and between genomes. The results show that synonymous codon usage is affected by two major factors: (i) the overall G+C content of the genome and (ii) growth at high temperature. This study focused on the relationship between synonymous codon usage and the ability to grow at high temperature. We have been able to eliminate both phylogenetic history and lateral gene transfer as possible explanations for the characteristic pattern of codon usage among the thermophiles. Thus, these results demonstrate a clear link between a particular pattern of codon usage and an external selective force.     

Regularities of context-dependent codon bias in eukaryotic genes

Nucleotides surrounding a codon influence the choice of this particular codon from among the group of possible synonymous codons. The strongest influence on codon usage arises from the nucleotide immediately following the codon and is known as the N₁ context. We studied the relative abundance of codons with N₁ contexts in genes from four eukaryotes for which the entire genomes have been sequenced: Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. For all the studied organisms it was found that 90% of the codons have a statistically significant N₁ context-dependent codon bias. The relative abundance of each codon with an N₁ context was compared with the relative abundance of the same 4mer oligonucleotide in the whole genome. This comparison showed that in about half of all cases the context-dependent codon bias could not be explained by the sequence composition of the genome. Ranking statistics were applied to compare context-dependent codon biases for codons from different synonymous groups. We found regularities in N₁ context-dependent codon bias with respect to the codon nucleotide composition. Codons with the same nucleotides in the second and third positions and the same N₁ context have a statistically significant correlation of their relative abundances.     

A thermodynamic theory of codon bias in viral genes

The relationship between degeneracy in the genetic code and the occurrence of a strong codon bias is examined, with particular reference to a group of viral genomes. The present paper shows how codon bias may have been imposed by thermodynamic considerations at the time the primitive DNA first formed in the primordial soup. Using a four-state Ising-like model with stacking interactions between successive base pairs, we show how primeval periodic DNA polymers could have arisen the remnants of which are still observed in codon biases today.     

Variation in G + C-content and codon choice: differences among synonymous codon groups in vertebrate genes.

The relationship between G + C-content and codon usage in genes of human, mus, rat, bovine and chicken nuclear genomes was investigated. Correlation and lineal regression analyses were carried out on plots that related the frequency of each codon within each synonymous codon group to the G + C-content of the coding sequence as a whole. Under GC pressure, in most of the quartet codon groups there is a preferential choice of the C-ending codon, except in leucine and valine codon groups where the choice of the G-ending codon is preferred. Among ducts, the choice of codons specifying phenylalanine and glutamate shows the strongest dependence on G + C-content. The relationship found between G + C-content and codon usage in these genomes correlate with taxonomic distance.     

Codon usage in the G+C-rich Streptomyces genome.

The codon usage (CU) patterns of 64 genes from the Gram+ prokaryotic genus Streptomyces were analysed. Despite the extremely high overall G+C content of the Streptomyces genome (estimated at 0.74), individual genes varied in G+C content from 0.610 to 0.797, and had third codon position G+C contents (GC3s) that varied from 0.764 to 0.983. The variation in GC3s explains a significant proportion of the variation in CU patterns. This is consistent with an evolutionary model of the Streptomyces genome where biased mutation pressure has led to a high average G+C content with random variation about the mean, although the variation observed is greater than that expected from a simple binomial model. The only gene in the sample that can be confidently predicted to be highly expressed, EF-Tu of Streptomyces coelicolor A3(2) (GC3s = 0.927), shows a preference for a third position C in several of the four codon families, and for CGY and GGY for Arg and Gly codons, respectively (Y = pyrimidine); similar CU patterns are found in highly expressed genes of the G+C-rich Micrococcus luteus genome. It thus appears that codon usage in Streptomyces is determined predominantly by mutation bias, with weak translational selection operating only in highly expressed genes. We discuss the possible consequences of the extreme codon bias of Streptomyces and consider how it may have evolved. A set of CU tables is provided for use with computer programs that locate protein-coding regions.     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.     

DNA G+C content of the third codon position and codon usage biases of human genes

The human genome, as in other eukaryotes, has a wide heterogeneity in the DNA base composition. The evolutionary basis for this heterogeneity has been unknown. A previous study of the human genome (846 genes analyzed) has shown that, in the major range of the G+C content in the third codon position (0.25-0.75), biases from the Parity Rule 2 (PR2) among the synonymous codons of the four-codon amino acids are similar except in the highest G+C range (Sueoka, N., 1999. Translation-coupled violation of Parity Rule 2 in human genes is not the cause of heterogeneity of the DNA G+C content of third codon position. Gene 238, 53-58.). PR2 is an intra-strand rule where A=T and G=C are expected when there are no biases between the two complementary strands of DNA in mutation and selection rates (substitution rates). In this study, 14,026 human genes were analyzed. In addition, the third codon positions of two-codon amino acids were analyzed. New results show the following: (a) The G+C contents of the third codon position of human genes are scattered in the G+C range of 0.22-0.96 in the third codon position. (b) The PR2 biases are similar in the range of 0.25-0.75, whereas, in the high G+C range (0.75-0.96; 13% of the genes), the PR2-bias fingerprints are different from those of the major range. (c) Unlike the PR2 biases, the G+C contents of the third codon position for both four-codon and two-codon amino acids are all correlated almost perfectly with the G+C content of the third codon position over the total G+C ranges. These results support the notion that the directional mutation pressure, rather than the directional selection pressure, is mainly responsible for the heterogeneity of the G+C content of the third codon position.     

The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias

Genes sequences from Escherichia coli, Salmonella typhimurium, and other members of the Enterobacteriaceae show a negative correlation between the degree of synonymous-codon usage bias and the rate of nucleotide substitution at synonymous sites. In particular, very highly expressed genes have very biased codon usage and accumulate synonymous substitutions very slowly. In contrast, there is little correlation between the degree of codon bias and the rate of protein evolution. It is concluded that both the rate of synonymous substitution and the degree of codon usage bias largely reflect the intensity of selection at the translational level. Because of the high variability among genes in rates of synonymous substitution, separate molecular clocks of synonymous substitution might be required for different genes.      

X-linked genes evolve higher codon bias in Drosophila and Caenorhabditis

Comparing patterns of molecular evolution between autosomes and sex chromosomes (such as X and W chromosomes) can provide insight into the forces underlying genome evolution. Here we investigate patterns of codon bias evolution on the X chromosome and autosomes in Drosophila and Caenorhabditis. We demonstrate that X-linked genes have significantly higher codon bias compared to autosomal genes in both Drosophila and Caenorhabditis. Furthermore, genes that become X-linked evolve higher codon bias gradually, over tens of millions of years. We provide several lines of evidence that this elevation in codon bias is due exclusively to their chromosomal location and not to any other property of X-linked genes. We present two possible explanations for these observations. One possibility is that natural selection is more efficient on the X chromosome due to effective haploidy of the X chromosomes in males and persistently low effective numbers of reproducing males compared to that of females. Alternatively, X-linked genes might experience stronger natural selection for higher codon bias as a result of maladaptive reduction of their dosage engendered by the loss of the Y-linked homologs.     

Synonymous codon usage in Cryptosporidium parvum: identification of two distinct trends among genes

The usage of alternative synonymous codons in the apicomplexan Cryptosporidium parvum has been investigated. A data set of 54 genes was analysed. Overall, A- and U-ending codons predominate, as expected in an A+T-rich genome. Two trends of codon usage variation among genes were identified using correspondence analysis. The primary trend is in the extent of usage of a subset of presumably translationally optimal codons, that are used at significantly higher frequencies in genes expected to be expressed at high levels. Fifteen of the 18 codons identified as optimal are more G+C-rich than the otherwise common codons, so that codon selection associated with translation opposes the general mutation bias. Among 40 genes with lower frequencies of these optimal codons, a secondary trend in G+C content was identified. In these genes, G+C content at synonymously variable third positions of codons is correlated with that in 5' and 3' flanking sequences, indicative of regional variation in G+C content, perhaps reflecting regional variation in mutational biases.     

Insulin sensitivity is not affected by mutation of codon 972 of the human IRS-1 gene

We investigated the relationship of codon 972 polymorphism of the insulin receptor substrate-1 (IRS-1) gene with insulin resistance in the Japanese population. Among 130 patients with type-2 diabetes mellitus (DM), we identified 6 who were heterozygous for the Gly972Arg mutation. Among 144 healthy subjects, 6 were heterozygous and 1 was homozygous for the mutation. A hyperinsulinemic euglycemic clamp study was performed in 3 of 6 diabetic patients with the heterozygous Gly972Arg mutation and in 60 without it. Both groups showed almost the same levels of insulin sensitivity (glucose infusion rate, GIR = 50.2 +/- 3.0 vs. 51.3 +/- 12.1 micromol/kg/min). Similarly, there was no difference in insulin sensitivity between healthy subjects with and without the mutation using the homeostasis model assessment (HOMA index = 1.14 +/- 0.50 vs. 1.02 +/- 0.63). The frequency of the Gly972Arg allele was not increased in diabetic patients compared with control subjects even in aged (>50 years old) or obese (BMI >/=25) subjects. Among healthy subjects, we identified a 25-year-old male with the homozygous Gly972Arg allele. He was slightly obese (BMI = 25.5) but showed relatively high insulin sensitivity, almost equal to that of healthy subjects without the mutation (GIR = 67.2 vs. 71.8 +/- 22.0 micromol/kg/min). Because the GIR in healthy subjects was significantly higher compared with that in type-2 DM patients, we speculate that another genetic or environmental factor producing a more deleterious effect on insulin sensitivity may exist in diabetic patients. We conclude that this gene abnormality does not play a role in the pathogenesis of insulin resistance and type-2 DM.     

Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid

While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an ‘accessory’ during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context.