Chloroplast DNA codon use: Evidence for selection at the psb A locus based on tRNA availability

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.     

Codon Usage Bias and tRNA Abundance in Drosophila

Codon usage bias of 1,117 Drosophila melanogaster genes, as well as fewer D. pseudoobscura and D. virilis genes, was examined from the perspective of relative abundance of isoaccepting tRNAs and their changes during development. We found that each amino acid contributes about equally and highly significantly to overall codon usage bias, with the exception of Asp which had very low contribution to overall bias. Asp was also the only amino acid that did not show a clear preference for one of its synonymous codons. Synonymous codon usage in Drosophila was consistent with ``optimal' codons deduced from the isoaccepting tRNA availability. Interestingly, amino acids whose major isoaccepting tRNAs change during development did not show as strong bias as those with developmentally unchanged tRNA pools. Asp is the only amino acid for which the major isoaccepting tRNAs change between larval and adult stages. We conclude that synonymous codon usage in Drosophila is well explained by tRNA availability and is probably influenced by developmental changes in relative abundance. Received: 5 December 1996 / Accepted: 14 June 1997     

Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage

To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

The Nonrandom Location of Synonymous Codons Suggests That Reading Frame-Independent Forces Have Patterned Codon Preferences

Biased codon usage is common in eukaryotic and prokaryotic genes. Evidence from Escherichia, Saccharomyces, and Drosophila indicates that it favors translational efficiency and accuracy. However, to date no functional advantages have been identified in the codon–anticodon interactions involving the most frequently used (preferred) codons. Here we present evidence that forces not related to the individual codon–anticodon interaction may be involved in determining which synonymous codons are preferred or avoided. We show that the ``off-frame' trinucleotide motif preferences inferrable from Drosophila coding regions are often in the same direction as Drosophila's ``in-frame' codon preferences, i.e., its codon usage. The off-frame preferences were inferred from the nonrandomness of the location of confamilial synonymous codons along coding regions—a pattern often described as a context dependence of nucleotide choice at synonymous positions or as codon-pair bias. We relied on randomizations of the location of confamilial codons that do not alter, and cannot be influenced by, the encoded amino acid sequences, codon usage, or base composition of the genes examined. The statistically significant congruency of in-frame and off-frame trinucleotide preferences suggests that the same kind of reading-frame-independent force(s) may also influence synonymous codon choice. These forces may have produced biases in codon usage that then led to the evolution of the translational advantages of these motifs as preferred codons. Under this scenario, tRNA pool size differences between preferred and nonpreferred codons initially were evolved to track the default overrepresentation of codons with preferred motifs. The motif preference hypothesis can explain the structuring of codon preferences and the similarities in the codon usages of distantly related organisms. Received: 10 November 1998 / Accepted: 23 February 1999     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.     

The Effects of Codon Context on In Vivo Translation Speed

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.     

Comparison of base composition and codon usage in insect mitochondrial genomes

Insects, the most biodiverse taxonomic group, have high AT content in their mitochondrial genomes. Although codon usage tends to be AT-rich, base composition and codon usage of mitochondrial genomes may vary among taxa. Thus, we compare base composition and codon usage patterns of 49 insect mitochondrial genomes. For protein coding genes, AT content is as high as 80% in the Hymenoptera and Lepidoptera and as low as 72% in the Orthopotera. The AT content is high at positions 1 and 3, but A content is low at position 2. A close correlation occurs between codon usage and tRNA abundance in nuclear genomes. Optimal codons can pair well with the antr codons of the most abundant tRNAs. One tRNA gene translates a synonymous codon family in vertebrate mitochondrial genomes and these tRNA anticodons can pair with optimal codons. However, optimal codons cannot pair with anticodons in mtDNA ofCochiiomyia hominivorax (Dipteral: CaLliphoridae). Ten optimal codons cannot pair with tRNA anticodons in all 49 insect mitochondrial genomes; non-optimal codon-anticodon usage is common and codon usage is not influenced by tRNA abundance.     

Synonymous codon ordering: a subtle but prevalent strategy of bacteria to improve translational efficiency

Background

In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes.

Methodology/Principal Findings

In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes.

Conclusions

Overall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.     

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.     

Distortion of tRNA upon near-cognate codon recognition on the ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

Growth rate dependence of transfer RNA abundance in Escherichia coli.

We have tested the predictions of a model that accounts for the codon preferences of bacteria in terms of a growth maximization strategy. According to this model the tRNA species cognate to minor and major codons should be regulated differently under different growth conditions: the isoacceptors cognate to major codons should increase at fast growth rates while those cognate to minor codons should decrease at fast growth rates. We have used a quantitative Northern blotting technique to measure the abundance of the methionine and the leucine isoacceptor families over growth rates ranging from 0.5 to 2.1 doublings per hour. Five tRNA species that are cognate to major codons (tRNA(eMet), tRNA(1fMet), tRNA(2fMet), tRNA(1Leu) and tRNA(3Leu) increase both as a relative fraction of total tRNA and in absolute concentration with increasing growth rates. Three tRNA species that are cognate to minor codons (tRNA(2Leu), tRNA(4Leu) and tRNA(5Leu) decrease as a relative fraction of total RNA and in absolute concentration with increasing growth rates. These data suggest that the abundances of groups of tRNA species are regulated in different ways, and that they are not regulated simply according to isoacceptor specificity. In particular, the data support the growth optimization model for codon bias.     

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.     

The second to last amino acid in the nascent peptide as a codon context determinant.

Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon. The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli. A 30-fold difference in readthrough of the UGA codon was observed. Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction. Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant. This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing. The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNA(Trp) as compared with a suppressor form of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.     

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.