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.     

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.     

Mutation bias is the driving force of codon usage in the Gallus gallus genome

Synonymous codons are used with different frequencies both among species and among genes within the same genome and are controlled by neutral processes (such as mutation and drift) as well as by selection. Up to now, a systematic examination of the codon usage for the chicken genome has not been performed. Here, we carried out a whole genome analysis of the chicken genome by the use of the relative synonymous codon usage (RSCU) method and identified 11 putative optimal codons, all of them ending with uracil (U), which is significantly departing from the pattern observed in other eukaryotes. Optimal codons in the chicken genome are most likely the ones corresponding to highly expressed transfer RNA (tRNAs) or tRNA gene copy numbers in the cell. Codon bias, measured as the frequency of optimal codons (Fop), is negatively correlated with the G + C content, recombination rate, but positively correlated with gene expression, protein length, gene length and intron length. The positive correlation between codon bias and protein, gene and intron length is quite different from other multi-cellular organism, as this trend has been only found in unicellular organisms. Our data displayed that regional G + C content explains a large proportion of the variance of codon bias in chicken. Stepwise selection model analyses indicate that G + C content of coding sequence is the most important factor for codon bias. It appears that variation in the G + C content of CDSs accounts for over 60% of the variation of codon bias. This study suggests that both mutation bias and selection contribute to codon bias. However, mutation bias is the driving force of the codon usage in the Gallus gallus genome. Our data also provide evidence that the negative correlation between codon bias and recombination rates in G. gallus is determined mostly by recombination-dependent mutational patterns.     

Synonymous codon usage analysis of the mycobacteriophage Bxz1 and its plating bacteria M. smegmatis: identification of highly and lowly expressed genes of Bxz1 and the possible function of its tRNA species

The extent of codon usage in the protein coding genes of the mycobacteriophage, Bxz1, and its plating bacteria, M. smegmatis, were determined, and it was observed that the codons ending with either G and / or C were predominant in both the organisms. Multivariate statistical analysis showed that in both organisms, the genes were separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. The second major explanatory axis differentiates the genes according to their genome type. A comparison of the relative synonymous codon usage between 20 highly- and 20 lowly expressed genes from Bxz1 identified 21 codons, which are statistically over represented in the former group of genes. Further analysis found that the Bxz1- specific tRNA species could recognize 13 out of the 21 over represented synonymous codons, which incorporated 13 amino acid residues preferentially into the highly expressed proteins of Bxz1. In contrast, seven amino acid residues were preferentially incorporated into the lowly expressed proteins by 10 other tRNA species of Bxz1. This analysis predicts for the first time that the Bxz1-specific tRNA species modulates the optimal expression of its proteins during development.     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

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.     

The repertoire of transfer RNA genes is tuned to codon usage bias in the genomes of Phytophthora sojae and Phytophthora ramorum

In all, 238 and 155 transfer (t)RNA genes were predicted from the genomes of Phytophthora sojae and P. ramorum, respectively. After omitting pseudogenes and undetermined types of tRNA genes, there remained 208 P. sojae tRNA genes and 140 P. ramorum tRNA genes. There were 45 types of tRNA genes, with distinct anticodons, in each species. Fourteen common anticodon types of tRNAs are missing altogether from the genome in the two species; however, these appear to be compensated by wobbling of other tRNA anticodons in a manner which is tied to the codon bias in Phytophthora genes. The most abundant tRNA class was arginine in both P. sojae and P. ramorum. A codon usage table was generated for these two organisms from a total of 9,803,525 codons in P. sojae and 7,496,598 codons in P. ramorum. The most abundant codon type detected from the codon usage tables was GAG (encoding glutamic acid), whereas the most numerous tRNA gene had a methionine anticodon (CAT). The correlation between the frequencies of tRNA genes and the codon frequencies in protein-coding genes was very low (0.12 in P. sojae and 0.19 in P. ramorum); however, the correlation between amino acid tRNA gene frequency and the corresponding amino acid codon frequency in P. sojae and P. ramorum was substantially higher (0.53 in P. sojae and 0.77 in P. ramorum). The codon usage frequencies of P. sojae and P ramorum were very strongly correlated (0.99), as were tRNA gene frequencies (0.77). Approximately 60% of orthologous tRNA gene pairs in P sojae and P. ramorum are located in regions that have conserved synteny in the two species.     

Codon bias and gene expression.

The frequencies with which individual synonymous codons are used to code their cognate amino acids is quite variable from genome to genome and within genomes, from gene to gene. One particularly well documented codon bias is that associated with highly expressed genes in bacteria as well as in yeast; this is the so-called major codon bias. Here, it is suggested that the major codon bias is not an arrangement for regulating individual gene expression. Instead, the data suggest that this codon bias, which is correlated with a corresponding bias of tRNA abundance, is a global arrangement for optimizing the growth efficiency of cells. On the practical side, it is suggested that heterologous gene expression is not as sensitive to codon bias as previously thought, but that it is quite sensitive to other characteristics of the heterologous gene.     

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.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Coevolution of codon usage and tRNA genes leads to alternative stable states of biased codon usage

The typical number of tRNA genes in bacterial genomes is around 50, but this number varies from under 30 to over 120. We argue that tRNA gene copy numbers evolve in response to translational selection. In rapidly multiplying organisms, the time spent in translation is a limiting factor in cell division; hence, it pays to duplicate tRNA genes, thereby increasing the concentration of tRNA molecules in the cell and speeding up translation. In slowly multiplying organisms, translation time is not a limiting factor, so the overall translational cost is minimized by reducing the tRNAs to only one copy of each required gene. Translational selection also causes a preference for codons that are most rapidly translated by the current tRNAs; hence, codon usage and tRNA gene content will coevolve to a state where each is adapted to the other. We show that there is often more than one stable coevolved state. This explains why different combinations of tRNAs and codon bias can exist for different amino acids in the same organism. We analyze a set of 80 complete bacterial genomes and show that the theory predicts many of the trends that are seen in these data.     

Impact of translational selection on codon usage bias in the archaeon Methanococcus maripaludis

Patterns of codon usage have been extensively studied among Bacteria and Eukaryotes, but there has been little investigation of species from the third domain of life, the Archaea. Here, we examine the nature of codon usage bias in a methanogenic archaeon, Methanococcus maripaludis. Genome-wide patterns of codon usage are dominated by a strong A + T bias, presumably largely reflecting mutation patterns. Nevertheless, there is variation among genes in the use of a subset of putatively translationally optimal codons, which is strongly correlated with gene expression level. In comparison with Bacteria such as Escherichia coli, the strength of selected codon usage bias in highly expressed genes in M. maripaludis seems surprisingly high given its moderate growth rate. However, the pattern of selected codon usage differs between M. maripaludis and E. coli: in the archaeon, strongly selected codon usage bias is largely restricted to twofold degenerate amino acids (AAs). Weaker bias among the codons for fourfold degenerate AAs is consistent with the small number of tRNA genes in the M. maripaludis genome.     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.     

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 usages in different gene classes of the Escherichia coli genome

A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of ‘highly expressed genes’ (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5′ third of genes deviate by more than a factor of two from those in the middle and 3′ thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100–300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function.     

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.     

Intragenic codon bias in a set of mouse and human genes

To better conceptualize the mechanism underlying the evolution of synonymous codons, we have analysed intragenic codon usage in chosen "regions" of some mouse and human genes. We divided a given gene into two regions: one consisting of a trinucleotide repeat (TNR) and the other consisting of the "rest of the coding region" (RCR). Usually, a TNR is composed of a repetitive single codon, which may reflect its frequency in a gene. In contrast, a non-random frequency of a codon in the RCR versus TNR (or vice versa) of a gene should indicate a bias for that codon within the TNR. We examined this scenario by comparing codon frequency between the RCR and the cognate TNR(s) for a set of human and mouse genes. A TNR length of six amino acids or more was used to identify genes from the Genbank database. Twenty nine human and twenty one mouse genes containing TNRs coding for nine different amino acid runs were identified. The ratio of codon frequency in a TNR versus the corresponding RCR was expressed as "fold change" which was also regarded as a measure of codon bias (defined as preferential use either in TNR or in RCR). Chi-square values were then determined from the distribution of codon frequency in a TNR vs. the cognate RCR. At p<0.001, 22% and 27%, respectively, of human and mouse TNRs showed codon bias. Greater than 40% of the TNRs (29 out of 69 in human, and 18 of 42 in mouse) showed codon bias at p<0.05. In addition, we identify eight single-codon TNRs in mouse and ten in human genes. Thus, our results show intragenic codon bias in both mouse and human genes expressed in diverse tissue types. Since our results are independent of the Codon Adaptation Index (CAI) and starvation CAI, and since the tRNA repertoire in a cell or in a tissue is constant, our data suggest that other constraints besides tRNA abundance played a role in creating intragenic codon bias in these genes.