Comparative Analysis of Codon Usage Bias and Codon Context Patterns between Dipteran and Hymenopteran Sequenced Genomes

Background

Codon bias is a phenomenon of non-uniform usage of codons whereas codon context generally refers to sequential pair of codons in a gene. Although genome sequencing of multiple species of dipteran and hymenopteran insects have been completed only a few of these species have been analyzed for codon usage bias.

Methods and Principal Findings

Here, we use bioinformatics approaches to analyze codon usage bias and codon context patterns in a genome-wide manner among 15 dipteran and 7 hymenopteran insect species. Results show that GAA is the most frequent codon in the dipteran species whereas GAG is the most frequent codon in the hymenopteran species. Data reveals that codons ending with C or G are frequently used in the dipteran genomes whereas codons ending with A or T are frequently used in the hymenopteran genomes. Synonymous codon usage orders (SCUO) vary within genomes in a pattern that seems to be distinct for each species. Based on comparison of 30 one-to-one orthologous genes among 17 species, the fruit fly Drosophila willistoni shows the least codon usage bias whereas the honey bee (Apis mellifera) shows the highest bias. Analysis of codon context patterns of these insects shows that specific codons are frequently used as the 3′- and 5′-context of start and stop codons, respectively.

Conclusions

Codon bias pattern is distinct between dipteran and hymenopteran insects. While codon bias is favored by high GC content of dipteran genomes, high AT content of genes favors biased usage of synonymous codons in the hymenopteran insects. Also, codon context patterns vary among these species largely according to their phylogeny.     

Identification of protein-coding sequences using the hybridization of 18S rRNA and mRNA during translation

We introduce a new approach in this article to distinguish protein-coding sequences from non-coding sequences utilizing a period-3, free energy signal that arises from the interactions of the 3′-terminal nucleotides of the 18S rRNA with mRNA. We extracted the special features of the amplitude and the phase of the period-3 signal in protein-coding regions, which is not found in non-coding regions, and used them to distinguish protein-coding sequences from non-coding sequences. We tested on all the experimental genes from Saccharomyces cerevisiae and Schizosaccharomyces pombe. The identification was consistent with the corresponding information from GenBank, and produced better performance compared to existing methods that use a period-3 signal. The primary tests on some fly, mouse and human genes suggests that our method is applicable to higher eukaryotic genes. The tests on pseudogenes indicated that most pseudogenes have no period-3 signal. Some exploration of the 3′-tail of 18S rRNA and pattern analysis of protein-coding sequences supported further our assumption that the 3′-tail of 18S rRNA has a role of synchronization throughout translation elongation process. This, in turn, can be utilized for the identification of protein-coding sequences.     

Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonymous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa, and L. lactis as their primary host. We use the concept of a “genome landscape,” which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such as GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.     

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 × 10⁻⁵ to a high of 6.9 × 10⁻⁴ per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.     

SCUMBLE: a method for systematic and accurate detection of codon usage bias by maximum likelihood estimation

The genetic code is degenerate—most amino acids can be encoded by from two to as many as six different codons. The synonymous codons are not used with equal frequency: not only are some codons favored over others, but also their usage can vary significantly from species to species and between different genes in the same organism. Known causes of codon bias include differences in mutation rates as well as selection pressure related to the expression level of a gene, but the standard analysis methods can account for only a fraction of the observed codon usage variation. We here introduce an explicit model of codon usage bias, inspired by statistical physics. Combining this model with a maximum likelihood approach, we are able to clearly identify different sources of bias in various genomes. We have applied the algorithm to Saccharomyces cerevisiae as well as 325 prokaryote genomes, and in most cases our model explains essentially all observed variance.     

A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting.     

Analysis of synonymous codon usage patterns in the genus Rhizobium

The codon usage patterns of rhizobia have received increasing attention. However, little information is available regarding the conserved features of the codon usage patterns in a typical rhizobial genus. The codon usage patterns of six completely sequenced strains belonging to the genus Rhizobium were analysed as model rhizobia in the present study. The relative neutrality plot showed that selection pressure played a role in codon usage in the genus Rhizobium. Spearman’s rank correlation analysis combined with correspondence analysis (COA) showed that the codon adaptation index and the effective number of codons (ENC) had strong correlation with the first axis of the COA, which indicated the important role of gene expression level and the ENC in the codon usage patterns in this genus. The relative synonymous codon usage of Cys codons had the strongest correlation with the second axis of the COA. Accordingly, the usage of Cys codons was another important factor that shaped the codon usage patterns in Rhizobium genomes and was a conserved feature of the genus. Moreover, the comparison of codon usage between highly and lowly expressed genes showed that 20 unique preferred codons were shared among Rhizobium genomes, revealing another conserved feature of the genus. This is the first report of the codon usage patterns in the genus Rhizobium.     

Selection on Codon Usage for Error Minimization at the Protein Level

Given the structure of the genetic code, synonymous codons differ in their capacity to minimize the effects of errors due to mutation or mistranslation. I suggest that this may lead, in protein-coding genes, to a preference for codons that minimize the impact of errors at the protein level. I develop a theoretical measure of error minimization for each codon, based on amino acid similarity. This measure is used to calculate the degree of error minimization for 82 genes of Drosophila melanogaster and 432 rodent genes and to study its relationship with CG content, the degree of codon usage bias, and the rate of nucleotide substitution. I show that (i) Drosophila and rodent genes tend to prefer codons that minimize errors; (ii) this cannot be merely the effect of mutation bias; (iii) the degree of error minimization is correlated with the degree of codon usage bias; (iv) the amino acids that contribute more to codon usage bias are the ones for which synonymous codons differ more in the capacity to minimize errors; and (v) the degree of error minimization is correlated with the rate of nonsynonymous substitution. These results suggest that natural selection for error minimization at the protein level plays a role in the evolution of coding sequences in Drosophila and rodents.Reviewing Editor: Dr. Massimo Di Giulio     

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.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

On Ribosome Load,Codon Bias and Protein Abundance

Different codons encoding the same amino acid are not used equally in protein-coding sequences. In bacteria, there is a bias towards codons with high translation rates. This bias is most pronounced in highly expressed proteins, but a recent study of synthetic GFP-coding sequences did not find a correlation between codon usage and GFP expression, suggesting that such correlation in natural sequences is not a simple property of translational mechanisms. Here, we investigate the effect of evolutionary forces on codon usage. The relation between codon bias and protein abundance is quantitatively analyzed based on the hypothesis that codon bias evolved to ensure the efficient usage of ribosomes, a precious commodity for fast growing cells. An explicit fitness landscape is formulated based on bacterial growth laws to relate protein abundance and ribosomal load. The model leads to a quantitative relation between codon bias and protein abundance, which accounts for a substantial part of the observed bias for E. coli. Moreover, by providing an evolutionary link, the ribosome load model resolves the apparent conflict between the observed relation of protein abundance and codon bias in natural sequences and the lack of such dependence in a synthetic gfp library. Finally, we show that the relation between codon usage and protein abundance can be used to predict protein abundance from genomic sequence data alone without adjustable parameters.     

Translational Features of Human Alpha 2b Interferon Production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5′ end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

Universal Pattern and Diverse Strengths of Successive Synonymous Codon Bias in Three Domains of Life, Particularly Among Prokaryotic Genomes

There has been significant progress in understanding the process of protein translation in recent years. One of the best examples is the discovery of usage bias in successive synonymous codons and its role in eukaryotic translation efficiency. We observed here a similar type of bias in the other two life domains, bacteria and archaea, although the bias strength was much smaller than in eukaryotes. Among 136 prokaryotic genomes, 98 were found to have significant bias from random use of successive synonymous codons with Z scores larger than three. Furthermore, significantly different bias strengths were found between prokaryotes grouped by various genomic or biochemical characteristics. Interestingly, the bias strength measured by a general Z score could be fitted well (R = 0.83, P < 10⁻¹⁵) by three genomic variables: genome size, G + C content, and tRNA gene number based on multiple linear regression. A different distribution of synonymous codon pairs between protein-coding genes and intergenic sequences suggests that bias is caused by translation selection. The present results indicate that protein translation is tuned by codon (pair) usage, and the intensity of the regulation is associated with genome size, tRNA gene number, and G + C content.     

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.     

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.     

Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae

In-frame stop codons normally signal termination during mRNA translation, but they can be read as ‘sense’ (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon. To identify novel contexts directing readthrough, under-represented 5′ and 3′ stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico. In contrast with the nucleotide bias 3′ of the stop codon, codon bias in the two codon positions 5′ of the termination codon showed no correlation with known effects on stop codon readthrough. However, individually, poor 5′ and 3′ context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration. A novel method analysing specific nucleotide combinations in the 3′ context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency. Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough. Thus selection against stop codon readthrough is a dominant force acting on 3′, but not on 5′, nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides. The approaches described can be employed to define potential readthrough contexts for any genome.     

Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5′ adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5′ and 3′ of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific ‘shifty’ codons.     

Analysis of synonymous codon usage patterns in different plant mitochondrial genomes

Codon usage in mitochondrial genome of the six different plants was analyzed to find general patterns of codon usage in plant mitochondrial genomes. The neutrality analysis indicated that the codon usage patterns of mitochondrial genes were more conserved in GC content and no correlation between GC12 and GC3. T and A ending codons were detected as the preferred codons in plant mitochondrial genomes. The Parity Rule 2 plot analysis showed that T was used more frequently than A. The EN_C-plot showed that although a majority of the points with low EN_C values were lying below the expected curve, a few genes lied on the expected curve. Correspondence analysis of relative synonymous codon usage yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggest that natural selection is likely to be playing a large role in codon usage bias in plant mitochondrial genomes, but not only natural selection but also other several factors are likely to be involved in determining the selective constraints on codon bias in plant mitochondrial genomes. Meantime, 1 codon (P. patens), 6 codons (Z. mays), 9 codons (T. aestivum), 15 codons (A. thaliana), 15 codons (M. polymorpha) and 15 codons (N. tabacum) were defined as the preferred codons of the six plant mitochondrial genomes.