Codon usage determines translation rate in Escherichia coli

We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure. We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged. The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other. The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures. The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second. We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons. In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol. In this way we could investigate the influence of mRNA structure on translation rate. This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments. We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase. Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate. In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.     

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.     

Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ''prefer'' optimal codons.

Codon usage patterns in the slime mould Dictyostelium discoideum have been re-examined (a total of 58 genes have been analysed). Considering the extreme A + T-richness of this genome (G + C = 22%), there is a surprising degree of codon usage variation among genes. For example, G + C content at silent sites varies from less than 10% to greater than 30%. It was previously suggested [Warrick, H.M. and Spudich, J.A. (1988) Nucleic Acids Res. 16: 6617-6635] that highly expressed genes contain fewer 'optimal' codons than genes expressed at lower levels. However, it appears that the optimal codons were misidentified. Multivariate statistical analysis shows that the greatest variation among genes is in relative usage of a particular subset of codons (about one per amino acid), many of which are C-ending. We have identified these as optimal codons, since (i) their frequency is positively correlated with gene expression level, and (ii) there is a strong mutation bias in this genome towards A and T nucleotides. Thus, codon usage in D. discoideum can be explained by a balance between the forces of mutational bias and translational selection.     

Codon usage in Plasmodium vivax nuclear genes.

Codon usage in Plasmodium vivax nuclear genes was analysed and compared with that in Plasmodium falciparum nuclear genes. Preferred codons were determined for P. vivax. Unlike P. falciparum, P. vivax genes are about 15% less A+T rich in the coding regions, with no obvious A+T bias at the third position of the codons. The amino-acid composition of P. vivax gene products is also different from that of P. falciparum. These results provide valuable information to facilitate gene cloning as well as expression and transfection studies for P. vivax.     

Valinomycin-induced cation transport in vesicles does not reflect the activity of K+ transport systems in Escherichia coli.

Transport systems for K+ in Escherichia coli are not detectable in membrane vesicles, but vesicles will take up K+ (and Rb+) in the presence of valinomycin. It is generally believed that valinomycin acts as a lipid-soluble cation carrier and that it does not interact with or activate cation transport systems. This view is challenged by Bhattacharyya et al. (Proc. Natl. Acad. Sci. USA 68:1448-1492, 1971), who reported reduced uptake in vesicles from E. coli mutants with K+ transport defects. We reexamined this question with some of the same mutants and were unable to confirm a correlation of valinomycin-induced vesicle transport with transport properties in intact cells. We found great variability in transport activity of vesicles from these E. coli K-12 strains and believe such variability as well as possible contamination with intact cells accounts for the earlier report. Our data do not support the idea that valinomycin-mediated transport in vesicles is related to physiological K+ transport systems.     

Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes.

AGA and AGG codons for arginine are the least used codons in Escherichia coli, which are encoded by a rare tRNA, the product of the dnaY gene. We examined the positions of arginine residues encoded by AGA/AGG codons in 678 E. coli proteins. It was found that AGA/AGG codons appear much more frequently within the first 25 codons. This tendency becomes more significant in those proteins containing only one AGA or AGG codon. Other minor codons such as CUA, UCA, AGU, ACA, GGA, CCC and AUA are also found to be preferentially used within the first 25 codons. The effects of the AGG codon on gene expression were examined by inserting one to five AGG codons after the 10th codon from the initiation codon of the lacZ gene. The production of beta-galactosidase decreased as more AGG codons were inserted. With five AGG codons, the production of beta-galactosidase (Gal-AGG5) completely ceased after a mid-log phase of cell growth. After 22 hr induction of the lacZ gene, the overall production of Gal-AGG5 was 11% of the control production (no insertion of arginine codons). When five CGU codons, the major arginine codon were inserted instead of AGG, the production of beta-galactosidase (Gal-CGU5) continued even after stationary phase and the overall production was 66% of the control. The negative effect of the AGG codons on the Gal-AGG5 production was found to be dependent upon the distance between the site of the AGG codons and the initiation codon. As the distance was increased by inserting extra sequences between the two codons, the production of Gal-AGG5 increased almost linearly up to 8 fold. From these results, we propose that the position of the minor codons in an mRNA plays an important role in the regulation of gene expression possibly by modulating the stability of the initiation complex for protein synthesis.     

Synonymous codon usage in Escherichia coli: selection for translational accuracy

In many organisms, selection acts on synonymous codons to improve translation. However, the precise basis of this selection remains unclear in the majority of species. Selection could be acting to maximize the speed of elongation, to minimize the costs of proofreading, or to maximize the accuracy of translation. Using several data sets, we find evidence that codon use in Escherichia coli is biased to reduce the costs of both missense and nonsense translational errors. Highly conserved sites and genes have higher codon bias than less conserved ones, and codon bias is positively correlated to gene length and production costs, both indicating selection against missense errors. Additionally, codon bias increases along the length of genes, indicating selection against nonsense errors. Doublet mutations or replacement substitutions do not explain our observations. The correlations remain when we control for expression level and for conflicting selection pressures at the start and end of genes. Considering each amino acid by itself confirms our results. We conclude that selection on synonymous codon use in E. coli is largely due to selection for translational accuracy, to reduce the costs of both missense and nonsense errors.     

Colicin V-treated Escherichia coli does not generate membrane potential.

Colicin V-treated Escherichia coli was inhibited in its capacity to carry out active transport of proline and was unable to generate a membrane potential. Colicin V also prevented membrane potential formation by isolated cytoplasmic membrane vesicles. We conclude that a primary effect of this colicin involves the cytoplasmic membrane as a target.     

Codon usage patterns among genes for lepidopteran hemolymph proteins

Patterns in codon usage were examined for the coding regions of the 23 known lepidopteran hemolymph proteins. Coding triplets are GC rich at the third position and a significant linear relationship between GC content of silent and nonsilent (replacement) sites was demonstrated. Intron GC content was significantly lower than in coding regions and no relationship between intron GC content and the same at silent and nonsilent sites was found. Though hemolymph proteins are all produced by the same tissue—fat body—significantly less bias was observed when all moth sequences were pooled than when sequences of the two major species were analyzed separately, as predicted by the genome hypothesis. In cases where no statistically significant bias was observed, polar or acidic basic amino acids were almost exclusively involved. Calculation of codon adaptation indices (CAI) was of limited value in quantifying the degree of codon bias and probably reflects the complexity of multicellular-organism life cycles and the changing patterns of gene expression over different developmental stages. Correspondence to: D.R. Frohlich     

Gradients in nucleotide and codon usage along Escherichia coli genes

The usage of codons and nucleotide combinations varies along genes and systematic variation causes gradients in usage. We have studied such gradients of nucleotides and nucleotide combinations and their immediate context in Escherichia coli. To distinguish mutational and selectional effects, the genes were subdivided into three groups with different codon usage bias and the gradients of nucleotide usage were studied in each group. Some combinations that can be associated with a propensity for processivity errors show strong negative gradients that become weaker in genes with low codon bias, consistent with a selection on translational efficiency. One of the strongest gradients is for third position G, which shows a pervasive positive gradient in usage in most contexts of surrounding bases.     

Codon usage and G+C content in Bradyrhizobium japonicum genes are not uniform

To date, the sequences of 45 Bradyrhizobium japonicum genes are known. This provides sufficient information to determine their codon usage and G+C content. Surprisingly, B. japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G+C content than genes not belonging to these two groups. Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes. The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj9 sequence which is an insertion sequence (IS)-like element. The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors. These were now corrected. The deduced gene product of ORF355 in RSRj9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes. The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements.     

Codon bias in Escherichia coli: the influence of codon context on mutation and selection.

The codon bias in Escherichia coli for all two-fold degenerate amino acids was studied as dependent on the context from the six bases in the nearest surrounding codons. By comparing the results in genes at different expression levels, effects that are due to differences in mutation rates can be distinguished from those that are due to selection. Selective effects on the codon bias is found mostly from the first neighbouring base in the 3'direction, while neighbouring bases further away influence mostly the mutational bias. In some cases it is also possible to identify specific molecular processes, repair or avoidance of frame shift, that lead to the context dependence of the bias.     

Codon usage in Pseudomonas aeruginosa.

We have generated a codon usage table for Pseudomonas aeruginosa. Codon usage in P. aeruginosa is extremely biased. In contrast to E. coli and yeast, P. aeruginosa preferentially uses those codons within a synonymous codon group with the strongest predicted codon-anticodon interaction. We were unable to correlate a particular codon usage pattern with predicted levels of mRNA expressivity. The choice of a third base reflects the high guanine plus cytosine content of the P. aeruginosa genome (67.2%) and cytosine is the preferred nucleotide for the third codon position.     

Codon usage in Entamoeba histolytica.

The codon usage of 10 E. histolytica genes comprising 4455 codons was analysed. The codon usage revealed an extremely biased use of synonymous codons with a preference for NNU (44%) and NNA (41.4%) codons. Codons CGG (arg), AGG (arg) and CCG (pro) were absent in the E. histolytica genes examined. The codon usage of E. histolytica resembled that of Plasmodium falciparum.     

Codon usage in the siliceous sponge Geodia cydonium: highly expressed genes in the simplest multicellular animals prefer C- and G-ending codons

Among a sample of 39 Geodia cydonium (Demospongiae, Porifera) genes, with an average G + C content of 51.2%, extensive structural heterogeneity and considerable variations in synonymous codon usage were found. The G + C content of coding sequences and G + C content at silent codon positions (GC3S) varied from 42.4 to 59.2% and from 35.6 to 76.5%, respectively. Correspondence analysis of 39 genes revealed that putative highly expressed genes preferentially use a limited subset of codons, which were therefore defined as preferred codons in G. cydonium . A total of 22 preferred codons for 18 amino acids with synonyms in codons were identified and they all (with one exception) end with C or G. Among these codons there are also C- and G-ending codons which were previously identified as codons optimal for translation in a variety of eukaryotes, including metazoans and plants. The bias in synonymous codon usage in putative highly expressed G. cydonium genes is moderate, indicating that these genes are not shaped under strong natural selection. We postulate that the preference for C- and G-ending codons was already established in the ancestor of all Metazoa, including also sponges. This ancestor most probably also had a G + C rich genome. The selection toward C- and G-ending codons has been largely conserved throughout eukaryote evolution; exceptions are, for example, mammals for which strong mutational biases caused switches from that rule.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.