Comparing expression level-dependent features in codon usage with protein abundance: an analysis of 'predictive proteomics'

Synonymous codon usage is a commonly used means for estimating gene expression levels of Escherichia coli genes and has also been used for predicting highly expressed genes for a number of prokaryotic genomes. By comparison of expression level-dependent features in codon usage with protein abundance data from two proteome studies of exponentially growing E. coli and Bacillus subtilis cells, we try to evaluate whether the implicit assumption of this approach can be confirmed with experimental data. Log-odds ratio scores are used to model differences in codon usage between highly expressed genes and genomic average. Using these, the strength and significance of expression level-dependent features in codon usage were determined for the genes of the Escherichia coli, Bacillus subtilis and Haemophilus influenzae genomes. The comparison of codon usage features with protein abundance data confirmed a relationship between these to be present, although exceptions to this, possibly related to functional context, were found. For species with expression level-dependent features in their codon usage, the applied methodology could be used to improve in silico simulations of the outcome of two-dimensional gel electrophoretic experiments.     

Unexpected correlations between gene expression and codon usage bias from microarray data for the whole Escherichia coli K-12 genome

Escherichia coli has long been regarded as a model organism in the study of codon usage bias (CUB). However, most studies in this organism regarding this topic have been computational or, when experimental, restricted to small datasets; particularly poor attention has been given to genes with low CUB. In this work, correspondence analysis on codon usage is used to classify E.coli genes into three groups, and the relationship between them and expression levels from microarray experiments is studied. These groups are: group 1, highly biased genes; group 2, moderately biased genes; and group 3, AT-rich genes with low CUB. It is shown that, surprisingly, there is a negative correlation between codon bias and expression levels for group 3 genes, i.e. genes with extremely low codon adaptation index (CAI) values are highly expressed, while group 2 show the lowest average expression levels and group 1 show the usual expected positive correlation between CAI and expression. This trend is maintained over all functional gene groups, seeming to contradict the E.coli-yeast paradigm on CUB. It is argued that these findings are still compatible with the mutation-selection balance hypothesis of codon usage and that E.coli genes form a dynamic system shaped by these factors.     

同义密码子携带多少蛋白质二级结构信息

应用信息论方法考察了大肠杆菌人两种生物的同义密码子用语和蛋白质二级结构的关联情况。研究结果表明：大肠杆菌和人的基因组中都存在着一些同义密码子明显携带有蛋白质二级结构信息,尽管这些信息量都很小;同义密码子与蛋白质二级结构的关联是种属特异性。     

The relationship among gene expression, folding free energy and codon usage bias in Escherichia coli

Taking advantage of microarray data in Escherichia coli genome, the relationship among mRNA expression levels, folding free energy and codon usage bias are investigated. Our results indicate that mRNA expression is correlated to the stability of mRNA secondary structure and the codon usage bias. The decrease of the stability of mRNA structure contributes to the increase of mRNA expression. There is a negative correlation between codon adaptation index (CAI) and mRNA expression in genes with less stable structure. The relationship between the stability of mRNA structure and mRNA half-life indicates the stability of mRNA structure is different from mRNA half-life.     

Intragenic spatial patterns of codon usage bias in prokaryotic and eukaryotic genomes

To study the roles of translational accuracy, translational efficiency, and the Hill-Robertson effect in codon usage bias, we studied the intragenic spatial distribution of synonymous codon usage bias in four prokaryotic (Escherichia coli, Bacillus subtilis, Sulfolobus tokodaii, and Thermotoga maritima) and two eukaryotic (Saccharomyces cerevisiae and Drosophila melanogaster) genomes. We generated supersequences at each codon position across genes in a genome and computed the overall bias at each codon position. By quantitatively evaluating the trend of spatial patterns using isotonic regression, we show that in yeast and prokaryotic genomes, codon usage bias increases along translational direction, which is consistent with purifying selection against nonsense errors. Fruit fly genes show a nearly symmetric M-shaped spatial pattern of codon usage bias, with less bias in the middle and both ends. The low codon usage bias in the middle region is best explained by interference (the Hill-Robertson effect) between selections at different codon positions. In both yeast and fruit fly, spatial patterns of codon usage bias are characteristically different from patterns of GC-content variations. Effect of expression level on the strength of codon usage bias is more conspicuous than its effect on the shape of the spatial distribution.     

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.     

The relationship between synonymous codon usage and protein structure in Escherichia coli and Homo sapiens

The role of silent position in the codon on the protein structure is an interesting and yet unclear problem. In this paper, 563 Homo sapiens genes and 417 Escherichia coli genes coding for proteins with four different folding types have been analyzed using variance analysis, a multivariate analysis method newly used in codon usage analysis, to find the correlation between amino acid composition, synonymous codon, and protein structure in different organisms. It has been found that in E. coli, both amino acid compositions in differently folded proteins and synonymous codon usage in different gene classes coding for differently folded proteins are significantly different. It was also found that only amino acid composition is different in different protein classes in H. sapiens. There is no universal correlation between synonymous codon usage and protein structure in these two different organisms. Further analysis has shown that GC content on the second codon position can distinguish coding genes for different folded proteins in both organisms.     

大肠杆菌mRNA编码区长度、形成二级结构倾向与密码子偏好性的关系

从GenBank获得大肠杆菌K-12MG1655株的全基因组序列,计算了与基因密码子偏好性相关的多个参数(Nc、CAI、GC、GC3s),对其mRNA编码区长度、形成二级结构倾向与密码子偏好性之间的关系进行了统计学分析,发现虽然翻译效率(包括翻译速度和翻译精度)是制约大肠杆菌高表达基因的密码子偏好性的主要因素,同时,mRNA编码区长度及其形成二级结构的倾向也是形成这种偏好性的不可忽略的原因,而且对偏好性有一定程度的削弱。另外对mRNA编码区形成二级结构倾向的生物学意义进行了讨论分析。     

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.     

杨树同义密码子用法的初步分析

杨树是世界上广泛栽培的重要造林树种之一,已经成为林木基因工程研究的模式植物。用杨树的314个蛋白编码基因,通过对应分析和ENC-plot分析探讨了若干重要因子对杨树密码子用法的效应。从分析结果中可以看出,在影响最大的第一条向量轴上,基因的坐标位置与该基因的表达水平(CAI)极显著负相关(r=-0.94**),其次是与GC3S和基因长度极显著相关(r=0.86**和r=-0.57**),说明基因表达水平高低是影响密码子发挥作用的主要因素,基因编码区碱基组成和基因长度次之。ENC-plot分析结果也证明了这一点。相对密码子使用值(RSCU)的计算结果表明,高表达基因强烈偏好以A或T结尾的密码子,并确定了TTA和ATA等10个密码子为杨树的主要偏爱密码子。将杨树的密码子使用频率与拟南芥、水稻、大肠杆菌和人等不同模式生物种比较后发现,杨树密码子的偏爱性与同为双子叶植物的拟南芥最为相似,与人和大肠杆菌之间的差异较大。     

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.     

Analysis of synonymous codon usage in Shigella flexneri 2a strain 301 and other Shigella and Escherichia coli strains

In this study, we analysed synonymous codon usage in Shigella flexneri 2a strain 301 (Sf301) and performed a comparative analysis of synonymous codon usage patterns in Sf301 and other strains of Shigella and Escherichia coli. Although there was a significant variety in codon usage bias among different Sf301 genes, there was a slight but observable codon usage bias that could primarily be attributable to mutational pressure and translational selection. In addition, the relative abundance of dinucleotides in Sf301 was observed to be independent of the overall base composition but was still caused by differential mutational pressure; this also shaped codon usage. By comparing the relative synonymous codon usage values across different Shigella and E. coli strains, we suggested that the synonymous codon usage pattern in the Shigella genomes was strain specific. This study represents a comprehensive analysis of Shigella codon usage patterns and provides a basic understanding of the mechanisms underlying codon usage bias.     

Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis.

Glycosyl hydrolase (GH) genes from Escherichia coli and Bacillus subtilis were used to search for cases of horizontal gene transfer. Such an event was inferred by G + C content, codon usage analysis, and a phylogenetic congruency test. The codon usage analysis used is a procedure based on a distance derived from a Pearson linear correlation coefficient determined from a pairwise codon usage comparison. The distances are then used to generate a distance-based tree with which we can define clusters and rapidly compare codon usage. Three genes (yagH from E. coli and xynA and xynB from B. subtilis) were determined to have arrived by horizontal gene transfer and were located in E. coli CP4-6 prophage, and B. subtilis prophages 6 and 5, respectively. In this study, we demonstrate that with codon usage analysis, the proposed horizontally transferred genes can be distinguished from highly expressed genes.     

Codon pairs in the genome of Escherichia coli

MOTIVATION: The effect of two neighboring codons (codon pairs) on gene expression is mediated via the interaction of their cognate tRNAs occupying the two functional ribosomal sites during the translation elongation step. For steric reasons it is reasonable to assume that not all combinations of codons and therefore of tRNAs are equally favorable when situated on the ribosome surface. Aiming of identifying preferential and rare codon pairs, we have determined the frequency of occurrence of all possible combinations of codon pairs in the entire genome of Escherichia coli (E.coli). RESULTS: The frequency of occurrence of the 3904 codon pairs comprising both sense:sense and sense:stop codon pairs in the full set of E.coli 4289 ORFs was found to vary from zero to 4913 times. For most of the pairs we have observed a significant difference between the real and statistically predicted frequency of occurrence. The analysis of 334 highly expressed and 303 poorly expressed E.coli genes showed that codon pair usage is different for the two gene categories. Using an especially defined criterion (Delta(REG)), the codon pairs are classified as 'hypothetically attenuating' (HAP) and 'hypothetically non-attenuating' (HNAP) and their possible effect on translation is discussed. AVAILABILITY: The program used in this study is available at http://www.bio21.bas.bg/codonpairs/     

Codon usage in bacteria: correlation with gene expressivity

The nucleic acid sequence bank now contains over 600 protein coding genes of which 107 are from prokaryotic organisms. Codon frequencies in each new prokaryotic gene are given. Analysis of genetic code usage in the 83 sequenced genes of the Escherichia coli genome (chromosome, transposons and plasmids) is presented, taking into account new data on gene expressivity and regulation as well as iso-tRNA specificity and cellular concentration. The codon composition of each gene is summarized using two indexes: one is based on the differential usage of iso-tRNA species during gene translation, the other on choice between Cytosine and Uracil for third base. A strong relationship between codon composition and mRNA expressivity is confirmed, even for genes transcribed in the same operon. The influence of codon use of peptide elongation rate and protein yield is discussed. Finally, the evolutionary aspect of codon selection in mRNA sequences is studied.     

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.     

Preferential use of A- and U-rich codons for Mycoplasma capricolum ribosomal proteins S8 and L6.

The nucleotide sequence of the 1.3 kilobase-pair DNA segment, which contains the genes for ribosomal proteins S8 and L6, and a part of L18 of Mycoplasma capricolum, has been determined and compared with the corresponding sequence in Escherichia coli (Cerretti et al., Nucl. Acids Res. 11, 2599, 1983). Identities of the predicted amino acid sequences of S8 and L6 between the two organisms are 54% and 42%, respectively. The A + T content of the M. capricolum genes is 71%, which is much higher than that of E. coli (49%). Comparisons of codon usage between the two organisms have revealed that M. capricolum preferentially uses A- and U-rich codons. More than 90% of the codon third positions and 57% of the first positions in M. capricolum is either A or U, whereas E. coli uses A or U for the third and the first positions at a frequency of 51% and 36%, respectively. The biased choice of the A- and U-rich codons in this organism has been also observed in the codon replacements for conservative amino acid substitutions between M. capricolum and E. coli. These facts suggest that the codon usage of M. capricolum is strongly influenced by the high A + T content of the genome.     

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.