鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach

Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position −3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions −3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.     

Alpha-thalassemia due to the deletion of nucleotides -2 and -3 preceding the AUG initiation codon affects translation efficiency both in vitro and in vivo.

We previously hypothesized that a 2 nucleotide deletion, causing a A-greater than C change at position -3 preceding the ATG initiation codon of alpha globin gene, reduced translation efficiency of alpha globin mRNA and was responsible for a form of alpha + thalassemia displayed by an Algerian patient. We presently show that this deletion leads to a 30-45% reduction in translation efficiency of synthetic alpha globin mRNA in rabbit reticulocyte lysate. In other experiments, we constructed alpha/G gamma hybrid globin genes in which the 3' end of normal or mutated alpha globin genes downstream to the ATG initiation codon was substituted by the 3' part of a G gamma globin gene. COS cells transfected with either of these 2 hybrid genes were shown to synthesize a similar amount of alpha/G gamma hybrid mRNAs but 50% less G gamma globin when transfected with the alpha/G gamma hybrid gene carrying the deletion. These results definitively establish that the 2 nucleotide deletion reduces translation efficiency by 30-50%. This contrasts with the 93% reduction induced by a similar A-greater than C change at position -3 in the different nucleotide context preceding the ATG codon of the rat preproinsulin gene.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.     

Proteomic analysis of the cyanobacterium Anabaena sp. strain PCC7120 with two-dimensional gel electrophoresis and amino-terminal sequencing

A protein-gene linkage map of the cyanobacterium Anabaena sp. strain PCC7120 was successfully constructed for 123 relatively abundant proteins. The total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the protein spots were determined. By comparing the determined amino-terminal sequences with the entire genome sequence, the putative translation initiation sites of 87 genes were successfully assigned on the genome. The elucidated sequence features surrounding the translation initiation sites were as follows: (1) GTG and TTG in addition to the ATG were used as rare initiation codons; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 51 initiation sites (58.6%); (3) the nucleotides at the two regions, from -35 to -33, and from -19 to -17 (relative to the first nucleotide in the initiation codon) were preferentially adenines or thymines; (4) the nucleotides at the region from -14 to -8 were preferentially purines; (5) the nucleotide at position -1 was biased towards non-guanine (96.6%); (6) the nucleotide at the position +5 was preferentially cytosine (63.2%). It was evident that removal of the translation initiator methionine was dependent on the side-chain bulkiness of the penultimate amino acid residue. The predicted putative signal peptide sequences were also indicated. Besides confirming the existence of many predicted proteins, the data will serve as a starting point for the study of signals important in post-translational processing and nucleotide sequences important in the initiation of translation.     

Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes.

An increasing number of cases where tri-nucleotide stop codons do not signal the termination of protein synthesis are being reported. In order to identify what constitutes an efficient stop signal, we analysed the region around natural stop codons in genes from a wide variety of eukaryotic species and gene families. Certain stop codons and nucleotides following stop codons are over-represented, and this pattern is accentuated in highly expressed genes. For example, the preferred signal for Saccharomyces cerevisiae and Drosophila melanogaster highly expressed genes is UAAG, and generally the signals UAA(A/G) and UGA(A/G) are preferred in eukaryotes. The GC% of the organism or DNA region can affect whether there is A or G in the second or fourth positions. We suggest therefore, that the stop codon and the nucleotide following it comprise a tetra-nucleotide stop signal. A model is proposed in which the polypeptide chain release factor, a protein, recognises this sequence, but will tolerate some substitution, particularly A to G in the second or third positions.     

Analysis of sequence patterns proximal to the stop codons in Oryza sativa

Abstract

A comprehensive analysis of sequence patterns around the stop codons was performed, by using more than 26,000 rice full-length cDNA sequences. Here it is shown that the bias was most outstanding at the position immediately before the stop codons (?1 codon), where the AAC codon was strongly preferred among ANC codons. Compared with other positions, the codon immediately after the stop codons (+1 codon) also displayed an apparent difference, and had a strong consensus for base A at the first, C at the second, and A at the third letters, respectively. Notably, the base biases at the positions directly downstream of the stop codons, such as the +4, +5 and +6 positions, were much stronger than other positions in the 3′-UTR region, suggesting that those base positions might act as an extended stop signal in the process of protein synthesis. Examination of the relationship between sequence pattern and gene expression level, assessed by CAI values and EST counting, revealed a tendency towards bigger base biases for highly expressed genes. It could be inferred that the translation stop signal is possibly involved in many sequence recognition elements other than the stop codons; highly expressed genes should hold strong sequence consensus around the stop codons for efficient translation termination.     

Sense codon in Escherichia coli are translated in context

The nucleotide frequencies 5' and 3' to the sense codons in highly and weakly expressed genes have been investigated by the chi-squares method. A comparison between the experimental and computer-generated random nucleotide sequences (in which each codon is substituted by a random synonymous one) was made. It was shown that the choice of a particular codon among the synonymous ones in a given position of the gene depends on the three nucleotides 3' and 5' adjacent to the codon in highly expressed genes (the triplet 3' and a single nucleotide 5' to the codons in weakly expressed genes). Concrete patterns for the preferable choice of synonymous codons depending on their contexts are presented. It is suggested that these constraints are related to the efficiency of messenger translation. The constraints on the amino acid sequences of encoded proteins also lead to statistically significant bases in nucleotide frequencies around the sense codons. The biological role of these constraints is discussed.     

Translational efficiency of a non-AUG initiation codon is significantly affected by its sequence context in yeast

Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions -3 to -1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (-3 to -1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated.     

Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon.

Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides. Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes. Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively.     

Analysis of Context Sequence Surrounding Translation Initiation Site from Complete Genome of Model Plants

Regions flanking the translation initiation site (TIS) are thought to play a crucial role in translation efficiency of mRNAs, but their exact sequence and evolution in eukaryotes are still a matter of debate. We investigated the context sequences in 20 nucleotides around the TIS in multi-cellular eukaryotes, with a focus on two model plants and a comparison to human. We identified consensus sequences aaaaaaa(A/G)(A/C)aAUGGcgaataata and ggcggc(g/c)(A/G)(A/C)(G/C)AUGGCggcggcgg for Arabidopsis thaliana and Oryza sativa, respectively. We observe strongly conserved G at position +4 and A or C at position -2; however, the exact nucleotide frequencies vary between the three organisms even at these conserved positions. The frequency of pyrimidines, which are considered sub optimum at position -3, is higher in both plants than in human. Arabidopsis is GC-depleted (AU-enriched) compared to both rice and human, and the enrichment is slightly stronger upstream than downstream of AUG. While both plants are similar though not identical in their variation of nucleotide frequencies, rice and human are more similar to each other than Arabidopsis and human. All three organisms display clear periodicity in A + G and C + U content when analyzing normalized frequencies. These findings suggest that, besides few highly conserved positions, overall structure of the context sequence plays a larger role in TIS recognition than the actual nucleotide frequencies.     

In Arabidopsis thaliana codon volatility scores reflect GC3 composition rather than selective pressure

ABSTRACT: BACKGROUND: Synonymous codon usage bias has typically been correlated with, and attributed to translational efficiency. However, there are other pressures on genomic sequence composition that can affect codon usage patterns such as mutational biases. This study provides an analysis of the codon usage patterns in Arabidopsis thaliana in relation to gene expression levels, codon volatility, mutational biases and selective pressures. RESULTS: We have performed synonymous codon usage and codon volatility analyses for all genes in the A. thaliana genome. In contrast to reports for species from other kingdoms, we find that neither codon usage nor volatility are correlated with selection pressure (as measured by dN/dS), nor with gene expression levels on a genome wide level. Our results show that codon volatility and usage are not synonymous, rather that they are correlated with the abundance of G and C at the third codon position (GC3). CONCLUSIONS: Our results indicate that while the A. thaliana genome shows evidence for synonymous codon usage bias, this is not related to the expression levels of its constituent genes. Neither codon volatility nor codon usage are correlated with expression levels or selective pressures but, because they are directly related to the composition of G and C at the third codon position, they are the result of mutational bias. Therefore, in A. thaliana codon volatility and usage do not result from selection for translation efficiency or protein functional shift as measured by positive selection.     

Selection of AUG initiation codons differs in plants and animals.

The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.     

G and T Nucleotide Contents Show Specie-Invariant Negative Correlation for All Three Codon Positions

Abstract

The nucleotide contents of the three codon positions show a number of statistical pairwise correlations, some of which are universal for all analysed genomes. Among the most prominent of these correlations are negative correlations between G and T contents found in genes of all species analysed. The pair A/C, which is complementary to G/T shows similar negative correlation in genes of most species. In the genes of several species including all mammalian genes studied, positive correlations between A and T contents, and G and C contents are found. Since these regularities are observed in all three codon positions they are connected with amino-acid content of proteins. Such correlations may origin from features of the mutation process or/and translation reading frame check. The well-known bias of the preference for G in the first codon position and its deficiency in the second is accompanied by opposite bias in T content. In the third codon position there is no general nucleotide preference, but its content is often biased with regard to GC content of the gene. G and T contents in this case are always shifted in the opposite directions Several ideas are drawn to explain this preference.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.