Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.     

Synonymous codon preferences in bacteriophage T4: a distinctive use of transfer RNAs from T4 and from its host Escherichia coli.

Codon usage data of bacteriophage T4 genes were compiled and synonymous codon preferences were investigated in comparison with tRNA availabilities in an infected cell. Since the genome of T4 is highly AT rich and its codon usage pattern is significantly different from that of its host Escherichia coli, certain codons of T4 genes need to be translated by appropriate host transfer RNAs present in minor amounts. To avoid this predicament, T4 phage seems to direct the synthesis of its own tRNA molecules and these phage tRNAs are suggested to supplement the host tRNA population with isoacceptors that are normally present in minor amounts. A positive correlation was found in that the frequency of E. coli optimal codons in T4 genes increases as the number of protein monomers per phage particle increases. A negative correlation was also found between the number of protein monomers per phage and the frequency of "T4 optimal codons", which are defined as those codons that are efficiently recognized by T4 tRNAs. From these observations it was proposed that tRNAs from the host are predominantly used for translation of highly expressed T4 genes while tRNAs from T4 tend to be used for translation of weakly expressed T4 genes. This distinctive tRNA-usage in T4 may be an optimization of translational efficiency, and an adjustment of T4-encoded tRNAs to the synonymous codon preferences, which are largely influenced by the high genomic AT-content, would have occurred during evolution.     

Modulation of lambda integrase synthesis by rare arginine tRNA

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.     

Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.     

Codon usage and gene expression.

The hypothesis that codon usage regulates gene expression at the level of translation is tested. Codon usage of Escherichia coli and phage lambda is compared by correspondence analysis, and the basis of this hypothesis is examined by connecting codon and tRNA distributions to polypeptide elongation kinetics. Both approaches indicate that if codon usage was random tRNA limitation would only affect the rarest tRNA species. General discrimination against their cognate codons indicates that polypeptide elongation rates are maintained constant. Thus, differences in expression of E. coli genes are not a consequence of their variable codon usage. The preference of codons recognized by the most abundant tRNAs in E. coli genes encoding abundant proteins is explained by a constraint on the cost of proof-reading.     

Functional significance of an evolutionarily conserved alanine (GCA) resume codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.     

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.     

Absence of effect of varying Thr-Leu codon pairs on protein synthesis in a T7 system

The over-represented threonine-leucine (Thr-Leu) codon pair ACG CUG has been previously reported to be inhibitory to translation compared to the synonymous under-represented Thr-Leu codon pair ACC CUG, in an E. coli system in which the codon pairs were located either 3 and 4, or 6 and 7, or 9 and 10 codons downstream from the initiating codon for the message [Irwin, B., Heck, J. D., and Hatfield, G. W. (1995) J. Biol. Chem. 270, 22801-22806]. In the work reported here, these synonymous codon pairs were tested in a T7 system, with the codon pairs located either 14 and 15, or 6 and 7 codons downstream from the AUG start codon. In contrast to the reported findings in the E. coli system, there was no difference found in translation between mRNAs containing the respective codon pairs in the T7 system. The reasons for the different findings remain unclear, but presumably are a consequence of differences between the E. coli and T7 systems used to assay gene expression. Nevertheless, as a result of this work, it appears that the effect of varying codon pairs reported in the E. coli system is not due to a difference in translational step times through the respective codon pairs, as previously proposed.     

Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

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     

Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.     

Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency

Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.     

Simple and efficient method for heterologous expression of clostridial proteins

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.     

In silico diagnosis of inherently inhibited gene expression focusing on initial codon combinations

The translation start site, immediately downstream from the start codon, is a dominant factor for gene expression in Escherichia coli. At present, no method exists to improve the expression level of cloned genes, since it remains difficult to find the best codon combination within the region. We determined the expression parameters that correspond to all sense codons within the first four codons using GFPuv which encodes a derivative of green fluorescent protein. Using a genetic algorithm (GA)-based computer program, these parameters were incorporated in a simple, static model for the prediction of translation efficiency, and optimized to the expression level for 137 randomly isolated GFPuv genes. The calculated initial translation index (ITI), also proven for the DsRed2 gene that encodes a red fluorescent protein, should provide a solution to overcome the gene expression problem in cloned genes whose expression is often inherently blocked at the translation process. The proposed method facilitates heterologous protein production in E. coli, the most commonly used host in biological and industrial fields.     

Constraints on codon context in Escherichia coli genes. Their possible role in modulating the efficiency of translation

The constraints on nucleotide sequences of highly and weakly expressed genes from Escherichia coli have been analysed and compared. Differences in synonymous codon spectra in highly and weakly expressed genes lead to different frequencies of nucleotides (in the first and third codon positions) and dinucleotides in the two groups of genes. It has been found that the choice of synonymous codons in highly expressed genes depends on the nucleotides adjacent to the codon. For example, lysine is preferably encoded by the AAA codon if guanosine is 3' to the lysine codon (AAA-G, P less than 10(-9)). And, on the contrary, AAG is used more often than AAA (P less than 0.001) if cytidine is 3' adjacent to lysine. Guanosine occurs more frequently than adenosine 5' to all the lysine codons (AAR, P less than 10(-5), i.e. NNG codons are preferred over the synonymous NNA codons 5' to the positions of lysine in the genes. The context effect was observed in nonsense and missense suppression experiments. Therefore, a hypothesis has been suggested that the efficiency of translation of some codons (for which the constraints on the adjacent nucleotides were found) can be modulated by the codon context. The rules for preferable synonymous codon choice in highly expressed genes depending on the nucleotides surrounding the codon are presented. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Decoding with the A:I wobble pair is inefficient.

tRNAs with inosine (I) in the first position read three codons ending in U, C and A. However, A-ending codons read with I are rarely used. In Escherichia coli, CGA/U/C are all read solely by tRNAICGArg. CGU and CGC are very common codons, but CGA is very rare. Three independent in vivo assays show that translation of CGA is relatively inefficient. In the first, nine tandem CGA cause a strong rho-mediated polar effect on expression of a lacZ reporter gene. The inhibition is made more extreme by a mutation in ribosomal protein S12 (rpsL), which indicates that ribosomal binding by tRNAICGArg is slow and/or unstable in the CGA cluster. The second assay, in which codons are substituted for the regulatory UGA of the RF2 frameshift, confirms that aa-tRNA selection is slow and/or unstable at CGA. In the third assay, CGA is found to be a poor 5' context for amber suppression, which suggests that an A:I base pair in the P site can interfere with translation of a codon in the A site. Two possible errors, frameshifting and premature termination by RF2, are not significant causes for inefficiency at CGA. It is concluded that the A:I pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare usage of codons read with the A:I base pair.     

Role of GC-biased mutation pressure on synonymous codon choice in Micrococcus luteus, a bacterium with a high genomic GC-content.

The GC (G + C, or G or C)-contents of codon silent positions in all two-codon sets and three codons AUY/A (IIe), and in most of the family boxes of Micrococcus luteus (genomic GC-content: 74%) are 95% to 100% in both the highly and weakly expressed genes. In some family boxes, there is a decrease in NNC codons and an increase in NNG codons from the highly expressed to weakly expressed genes without apparent involvement of NNU and NNA codons. From these observations, we conclude that the selective use of synonymous codons in M. luteus may be largely determined by GC-biased mutation pressure and that in the highly expressed genes tRNAs would act as a weak selection pressure in some family boxes. Available data suggest that the effect of selection pressure by tRNAs on the synonymous codon choice becomes more apparent in the highly expressed genes in eubacteria with intermediate GC-contents such as Escherichia coli and Bacillus subtilis, and that the U/C ratio of the codon third positions in NNU/C-type two-codon sets in the weakly expressed genes would represent the approximate magnitude of directional mutation pressure throughout eubacteria.     

Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.