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.     

Statistical analysis of the 5' untranslated region of human mRNA using "Oligo-Capped" cDNA libraries

We constructed 34 types of human "full-length enriched" and "5'-end enriched" cDNA libraries based on the "Oligo-Capping" method. We randomly picked and sequenced 10,000 clones from these libraries. BLAST analysis showed that about 50% of the cDNAs were identical to known genes. Among them, we selected 954 species of cDNA that should represent the entire sequence from the mRNA start sites. Compared with previously reported sequences, they were on average 45 bp longer in the 5'-end. Using these cDNA data, we statistically analyzed the sequence features of the 5'UTR. The average length of the 5'UTR was 125 bp, and there was little correlation with the corresponding mRNA length (correlation coefficient = 0.26). Of the 954 species of 5'UTR, 459 contained no in-frame terminator codon, which is against the common belief. Two hundred seventy-eight species contained at least one ATG codon upstream of the initiator ATG codon. We identified 569 upstream ATGs, in total, 63% of which adequately satisfied Kozak's criteria. These findings are contrary to the typical translation initiation model, which states that translation is initiated from the "first" ATG codon.     

Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes

By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.     

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.     

The sequence flanking translational initiation site in protozoa.

Nucleotide sequences flanking the translational initiation site were compiled from protozoan nuclear genes and were compared in every protozoan group. The entire 5'-untranslated sequences were very rich in A- and T-residues, but poor in G- and C-residues in most protozoan genes except for the flagellated ones. The sequence AAAAATTTTTAAAATTTAAAATGANAT emerged as a consensus sequence flanking the initiation site in the major protozoan group, although the sequences upstream from -4 (four nucleotides upstream from the ATG codon) were divergent among ciliates, sarcodinians, and sporozoans. On the other hand, the consensus sequence for flagellates was revealed to be a simple feature. Only the nucleotide position -3 was occupied with a high frequency of A-residue, in other positions it appeared randomly. These facts suggest that the strong preference for A-residue at the position -3 is a universal feature in nuclear genes for all eukaryotes.     

Quantification analysis of translation initiation signal in vertebrate mRNAs: effect of nucleotides at positions +4(-)+6 upon efficiency of translation initiation.

Concerning the translation initiation signal in vertebrate mRNAs, a consensus, sequence, (GCC)GCC(A or G) CCATGG, has been proposed, but actual initiation sequences differ from it in a greater or lesser degree. Kozak monitored selection by ribosomes of the first versus second ATG codons as a function of mutations introduced at positions +4, and +6 of the first ATG codon. Codons possessing G at +4 strongly enhanced selection of the first ATG codon. However, ATG codon recognition was unaffected by most mutations in positions and +6. These data were well understood by our quantification analysis.     

Context sequences of translation initiation codon in plants

In this survey of 5074 plant genes for their AUG context sequences, purines are present at the _3 and +4 positions in about 80% of the sequences. Although this observation is similar to the vertebrate consensus sequence, the number of plant mRNAs with purines at the _3 position is lower and at the +4 position is higher than reported for vertebrate mRNAs. Higher plants have an AC-rich consensus sequence, caA(A/C)aAUGGCg as a context of translation initiator codon. Between the two major groups of angiosperms, the context of the AUG codon in dicot mRNAs is aaA(A/C)aAUGGCu which is similar to the higher-plant consensus but monocot mRNAs have c(a/c)(A/G)(A/C)cAUGGCG as a consensus which exhibits an overall similarity with the vertebrate consensus. The experimental evidence regarding the importance of the AUG context in plants is discussed.     

Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes.

Sequences flanking the initiator codon in eukaryotic mRNAs are not random. Out of 153 messages examined, 151 have either a purine in position -3, or a G in position +4, or both. Thus, [A/G]XXAUGG emerges as the favored sequence for eukaryotic initiation sites. Nucleotides flanking nonfunctional AUG triplets, which occur in the 5'-noncoding region of a few eukaryotic messages, are different from those found at most functional sites. Whereas most authentic initiator codons are preceded by a purine (usually A) in position -3, most nonfunctional AUGs have a pyrimidine in that position. The observed asymmetry suggests that purines in positions -3 and +4 might facilitate recognition of the AUG condon during formation of initiation complexes. To test this idea, in vitro binding studies were carried out with 32P-labeled oligonucleotides. Binding of AUG-containing oligonucleotides to wheat germ ribosomes was significantly enhanced by placing a purine in position -3 or +4. The scanning model, which postulates that 40S ribosomal subunits attach at the 5'-end of a message and migrate down to the AUG codon, is discussed in light of these new observations. A modified version of the scanning mechanism is proposed.     

At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells

Sequences flanking the AUG initiator codon influence its recognition by eukaryotic ribosomes. From a comparison of several hundred mRNA sequences, CCA/GCCAUGG emerged as the consensus sequence for initiation in higher eukaryotes. Systematic mutagenesis of a cloned preproinsulin gene confirmed the facilitating effect of A or G in position -3 (i.e. 3 nucleotides upstream from the AUG codon), C in positions -1 and -2, and G immediately following the AUG codon. The analysis of a new set of mutants now reveals that sequences slightly farther upstream are also influential, the optimal context for initiation being (GCC)GCCA/GCCAUGG. Possible mechanistic implications of the repeating GCC motif are discussed.     

Organization and expression of the E4 region of adenovirus 2.

The E4 region of Adenovirus 2 is a leftward transcribed part of the viral genome. Its nucleotide sequence has already been analysed. From this sequence several open reading frames were defined, which could be used in the coding of the E4 proteins. Using S1 digestion of mRNA-DNA hybrids a precise mapping of donor and acceptor sites was done. Their use in various combinations allows the synthesis of mRNAs, able to direct the synthesis of at least 7 polypeptides, ranging in size from 9K to 34K. Comparison of the sequences of the different acceptor sites indicates that they all conform to the consensus sequence. Analysis of the ATG surrounding sequence shows that initiator ATG may be positively selected according to Kozak's rule.     

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.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.