Efficient translation initiation dictates codon usage at gene start

The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein‐coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.     

Translation elongation can control translation initiation on eukaryotic mRNAs

Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.     

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure

The FGLamide allatostatins (ASTs) are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders. They also show myomodulatory activity. FGLamide AST nucleotide frequencies and codon bias were investigated with respect to possible effects on mRNA secondary structure. 367 putative FGLamide ASTs and their potential endoproteolytic cleavage sites were identified from 40 species of crustaceans, chelicerates and insects. Among these, 55% comprised only 11 amino acids. An FGLamide AST consensus was identified to be (X)_1→16Y(S/A/N/G)FGLGKR, with a strong bias for the codons UUU encoding for Phe and AAA for Lys, which can form strong Watson-Crick pairing in all peptides analyzed. The physical distance between these codons favor a loop structure from Ser/Ala-Phe to Lys-Arg. Other loop and hairpin loops were also inferred from the codon frequencies in the N-terminal motif, and the first amino acids from the C-terminal motif, or the dibasic potential endoproteolytic cleavage site. Our results indicate that nucleotide frequencies and codon usage bias in FGLamide ASTs tend to favor mRNA folds in the codon sequence in the C-terminal active peptide core and at the dibasic potential endoproteolytic cleavage site.     

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5 leader region of brome mosaic virus (BMV) RNA4 and the goat pre--lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.     

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation I_TE is formulated to facilitate studies on the efficiency and evolution of the translation machinery.     

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.     

Analysis of synonymous codon usage bias in Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexuallytransmitted diseases,and are associated with cardiovascular diseases.The analysis of codon usage mayimprove our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of targetgenes to improve their expression for gene therapy.Here,we analyzed the codon usage of C.muridarum,C.trachomatis(here indicating biovar trachoma and LGV),C.pneumoniae,and C.psittaci using the codonusage database and the CUSP(Create a codon usage table)program of EMBOSS(The European MolecularBiology Open Software Suite).The results show that the four genomes have similar codon usage patterns,with a strong bias towards the codons with A and T at the third codon position.Compared with Homosapiens,the four chlamydial species show discordant seven or eight preferred codons.The ENC(effectivenumber of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained bythe G+C content,while translational selection and gene length exert relatively weaker influences.Moreover,mutational pressure appears to be the major determinant of the codon usage variation among the chlamydialgenes.In addition,we compared the codon preferences of C.trachomatis with those of E.coli,yeast,adenovirus and Homo sapiens.There are 23 codons showing distinct usage differences between C.trachomatisand E.coli,24 between C.trachomatis and adenovirus,21 between C.trachomatis and Homo sapiens,butonly six codons between C.trachomatis and yeast.Therefore,the yeast system may be more suitable for theexpression of chlamydial genes.Finally,we compared the codon preferences of C.trachomatis with those ofsix eukaryotes,eight prokaryotes and 23 viruses.There is a strong positive correlation between the differ-ences in coding GC content and the variations in codon bias(r=0.905,P<0,001).We conclude that thevariation of codon bias between C.trachomatis and other organisms is much less influenced by phylogeneticlineage and primarily determined by the extent of disparities in GC content.     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻(Oryza rufipogon Griff.)线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻(O. sativa L.)的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示:普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数(Nc)分布于45.32～61.00之间,其密码子偏性较弱; Nc值仅与GC_3呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数(CBI)和最优密码子使用频率(Fop)的相关性均达到显著水平;而GC_3和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度(RSCU)、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻(O. sativa L. subsp. japonica Kato)和籼稻(O. sativa L. subsp.indica Kato)内具有同质性。     

New insights into the interplay between codon bias determinants in plants

Codon bias is the non-random use of synonymous codons, a phenomenon that has been observed in species as diverse as bacteria, plants and mammals. The preferential use of particular synonymous codons may reflect neutral mechanisms (e.g. mutational bias, G|C-biased gene conversion, genetic drift) and/or selection for mRNA stability, translational efficiency and accuracy. The extent to which these different factors influence codon usage is unknown, so we dissected the contribution of mutational bias and selection towards codon bias in genes from 15 eudicots, 4 monocots and 2 mosses. We analysed the frequency of mononucleotides, dinucleotides and trinucleotides and investigated whether the compositional genomic background could account for the observed codon usage profiles. Neutral forces such as mutational pressure and G|C-biased gene conversion appeared to underlie most of the observed codon bias, although there was also evidence for the selection of optimal translational efficiency and mRNA folding. Our data confirmed the compositional differences between monocots and dicots, with the former featuring in general a lower background compositional bias but a higher overall codon bias.     

Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms

Gene expression is known to correlate with the degree of codon bias in many unicellular organisms. However, such a correlation is not observed in some organisms. It was demonstrated that inverted complementary repeats within coding DNA sequences (ORFs) should be considered for proper estimation of the translation efficiency because they can form secondary structures that obstruct ribosome movement. A program was developed for estimating the potential expression of ORFs in unicellular organisms on the basis of their genome sequences. The program computes the elongation efficiency index (EEI) and takes into account three key factors: codon bias, the average number of inverted complementary repeats, and the free energies of potential stem-loop structures formed by these repeats. The influence of these factors on translation was numerically estimated. Their optimal ratio was computed for each organism. EEIs of 384 unicellular organisms (351 bacteria, 28 archaea, and 5 eukaryotes) were computed using the annotated genomes available from GenBank. Five potential evolutionary strategies of translational optimization were determined in the organisms studied. A considerable difference in preferential translational strategies was observed between bacteria and archaea. Significant correlations between EEIs and gene expression levels were shown for two species (Saccharomyces cerevisiae and Helicobacter pylori), using the available microarray data. The method allows the numerical estimation of the translation efficiency of an ORF and optimization of the nucleotide composition of heterologous genes in specified unicellular organisms. The program is available at http://wwwmgs.bionet.nsc.ru/mgs/programs/eei-calculator.     

A cross talk between codon usage bias in human oncogenes

Background: Oncogenes are the genes that have the potential to induce cancer. The extent and origin of codon usage bias is an important indicator of the forces shaping genome evolution in living organisms. Results: We observed moderate correlations between gene expression as measured by CAI and GC content at any codon site. The findings of our results showed that there is a significant positive correlation (Spearman''s r= 0.45, P<0.01) between GC content at first and second codon position with that of third codon position. Further, striking negative correlation (r = -0.771, P < 0.01) between ENC with the GC3s values of each gene and positive correlation (r=0.644, P<0.01) in between CAI and ENC was also observed. Conclusions: The mutation pressure is the major determining factor in shaping the codon usage pattern of oncogenes rather than natural selection since its effects are present at all codon positions. The results revealed that codon usage bias determines the level of oncogene expression in human. Highly expressed oncogenes had rich GC contents with high degree of codon usage bias.     

敏捷气热菌起始密码子侧翼序列与密码子偏好及基因长度相关性研究

好氧超嗜热古菌敏捷气热菌 (Aeropyrumpernix)mRNA中起始密码子AUG侧翼序列的保守性以及它与密码子使用偏好及基因长度之间具有相关性。AUG侧翼序列的保守性由M1(1)值表示 ,AUG侧翼序列对翻译起始的有效性由AUGCAI值表示。研究表明 :高表达和低表达基因的 - 2 0位到 13位中某些位点的保守性存在差异 ,其中高表达基因的 - 4位和 - 3位可能与其高表达的特性有关 ;在A .pernix中一个普遍的趋势是 :较短的基因有较高的表达效率 ,较长的基因的表达效率较低。与仅使用密码子偏好相比 ,将AUGCAI值引入到研究古菌在翻译水平上的自然选择更准确、更具有广泛适应性     

Evidence for a trade-off between translational efficiency and splicing regulation in determining synonymous codon usage in Drosophila melanogaster

In Drosophila melanogaster, synonymous codons corresponding to the most abundant cognate tRNAs are used more frequently, especially in highly expressed genes. Increased use of such "optimal" codons is considered an adaptation for translational efficiency. Need it always be the case that selection should favor the use of a translationally optimal codon? Here, we investigate one possible confounding factor, namely, the need to specify information in exons necessary to enable correct splicing. As expected from such a model, in Drosophila many codons show different usage near intron-exon boundaries versus exon core regions. However, this finding is in principle also consistent with Hill-Robertson effects modulating usage of translationally optimal codons. However, several results support the splice model over the translational selection model: 1) the trends in codon usage are strikingly similar to those in mammals in which codon usage near boundaries correlates with abundance in exonic splice enhancers (ESEs), 2) codons preferred near boundaries tend to be enriched for A and avoid C (conversely those avoided near boundaries prefer C rather than A), as expected were ESEs involved, and 3) codons preferred near boundaries are typically not translationally optimal. We conclude that usage of translationally optimal codons usage is compromised in the vicinity of splice junctions in intron-containing genes, to the effect that we observe higher levels of usage of translationally optimal codons at the center of exons. On the gene level, however, controlling for known correlates of codon bias, the impact on codon usage patterns is quantitatively small. These results have implications for inferring aspects of the mechanism of splicing given nothing more than a well-annotated genome.     

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.     

Codon bias signatures, organization of microorganisms in codon space, and lifestyle

New and simple numerical criteria based on a codon adaptation index are applied to the complete genomic sequences of 80 Eubacteria and 16 Archaea, to infer weak and strong genome tendencies toward content bias, translational bias, and strand bias. These criteria can be applied to all microbial genomes, even those for which little biological information is known, and a codon bias signature, that is the collection of strong biases displayed by a genome, can be automatically derived. A codon bias space, where genomes are identified by their preferred codons, is proposed as a novel formal framework to interpret genomic relationships. Principal component analysis confirms that although GC content has a dominant effect on codon bias space, thermophilic and mesophilic species can be identified and separated by codon preferences. Two more examples concerning lifestyle are studied with linear discriminant analysis: suitable separating functions characterized by sets of preferred codons are provided to discriminate: translationally biased (hyper)thermophiles from mesophiles, and organisms with different respiratory characteristics, aerobic, anaerobic, facultative aerobic and facultative anaerobic. These results suggest that codon bias space might reflect the geometry of a prokaryotic "physiology space." Evolutionary perspectives are noted, numerical criteria and distances among organisms are validated on known cases, and various results and predictions are discussed both on methodological and biological grounds.     

Study of codon bias perspective of fungal xylanase gene by multivariate analysis

Fungal xylanases has important applications in food, baking, pulp and paper industries in addition to various other industries. Xylanases are produced extensively by both bacterial and fungal sources and has tremendous potential of being active at extremes of temperature and pH. In the present study an effort has been made to explore the codon bias perspective of this potential enzyme using bioinformatics tools. Multivariate analysis has been used as a tool to study codon bias perspectives of xylanases. It was further observed that the codon usage of xylanases genes from different fungal sources is not similar and to reveal this phenomenon the relative synonymous codon usage (RSCU) and base composition variation in fungal xylanase genes were also studied. The codon biasing data like GC content at third position (GC_3S), effective codon number (N_C), codon adaptive index (CAI) were further analyzed with statistical softwares like Sigma1plot 9.0 and Systat 11.0. Furthermore, study of translation selection was also performed to verify the influences of codon usage variation among the 94 xylanase genes. In the present study xylanase gene from 12 organisms were analyzed and codon usages of all xylanases from each organism were compared separately. Analysis indicates biased codon among all 12 fungi taken for study with Aspergillus nidulans, Chaetomium globosum, Aspergillus terreus and Aspergillus clavatus showing maximum biasing. N_C plot and correspondence analysis on relative synonymous codon usage indicate that mutation bias and translation selection influences codon usage variation in fungal xylanase gene. To reveal the relative synonymous codon usage and base composition variation in xylanase, 94 genes from 12 fungi were used as model system.     

Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system

The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system. We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR). The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed. Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency. Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation. Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).