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.     

Sequence and structural features associated with translational initiator regions in yeast--a review

We have compared the translational initiator regions of 131 yeast genes. 95% utilize the first AUG from the 5' end of the message as the start codon for translation. Yeast leader regions in general are rich in adenine nucleotides (nt), have an average length of 52 nt, and are void of significant secondary structure. Sequences immediately adjacent to AUG start codons are preferred, however, the bias in nucleotide distribution (5'-A-YAA-UAAUGUCU-3') does not reflect a higher eukaryotic consensus (5'-CACCAUGG-3') with the exception of an adenine nucleotide preference at the -3 position. A minority of yeast mRNAs that contain AUG codons in the leader region that do not serve as the start codon for the primary gene product differ from the majority of mRNAs by one or more of these general properties. This analysis appears to indicate that basic features associated with yeast leader regions are consistent with a general mechanism of initiation of protein synthesis in eukaryotes, as proposed by the ribosomal 'scanning' model, but perhaps only basic features associated with ribosomal recognition of an AUG start codon are intact.     

AUG codons at the beginning of protein coding sequences are frequent in eukaryotic mRNAs with a suboptimal start codon context

MOTIVATION: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. However, mRNAs with a suboptimal context of start AUG codon are relatively abundant. It is likely that at least some mRNAs with suboptimal start codon context contain the other signals providing additional information for efficient AUG recognition. RESULTS: Frequency of AUG codons at the beginning of the coding part of eukaryotic mRNAs was analyzed in relation to the context of translation start codon. It was found that the observed downstream AUG content in the mRNAs with optimal start codon context was close to the expected value, whereas it was significantly higher in the mRNAs with a suboptimal context. It is likely that downstream AUG codons can often be utilized as additional start sites to increase translation rate of mRNAs with a suboptimal context of the annotated start codon and many eukaryotic proteins can be characterized by some N-end heterogeneity.     

Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.     

Emerging links between initiation of translation and human diseases

Some diseases are caused by mutations that perturb the initiation step of translation by changing the context around the AUG(START) codon or introducing upstream AUG codons. The scanning mechanism provides a framework for understanding the effects of these and other structural changes in mRNAs derived from oncogenes, tumor suppressor genes, and other key regulatory genes. In mRNAs from mutated as well as normal genes, translation sometimes initiates from an internal AUG codon. Sanctioned mechanisms that allow this, including leaky scanning and reinitiation, are discussed. Thrombopoietin mRNA is an example in which translation normally initiates from an internal position via an inefficient reinitiation mechanism. Mutations that restructure this mRNA in ways that elevate production of thrombopoietin cause hereditary thrombocythemia, demonstrating that some mRNAs are designed deliberately with upstream AUG codons to preclude efficient translation and thus to prevent harmful overproduction of potent proteins. While upstream AUG codons in certain mRNAs thus play an important regulatory role, the frequency of upstream AUG codons tends to be exaggerated when cDNA sequences are compiled and analyzed. Because the discovery of mutations that perturb translation usually begins with cDNA analysis, some misunderstandings vis-a-vis the interpretation of cDNA sequences are discussed.     

Positions +5 and +6 can be major determinants of the efficiency of non-AUG initiation codons for protein synthesis.

Only rarely do GUG (or CUG or ACG) codons which precede the 5'-proximal AUG function as initiators of protein synthesis, even when they are within a context that contains a purine at position -3 and a G at +4. For example, the upstream GUG of the human parainfluenza virus type 1 (hPIV1) P gene is initiated by ribosomes at high frequency, whereas a seemingly similar GUG codon in the hPIV3 P gene is not used at all. We have examined the reasons for this by expressing chimeric hPIV3/hPIV1 mRNAs, both in vivo and in vitro. A major determinant for efficient GUG utilization was located downstream of the GUG, but this did not appear to be involved in the formation of secondary structure. Rather, the sequence immediately downstream was found to be critical; this determinant was mapped to positions +5 and +6. GUG could be used efficiently for ribosomal initiation when the second codon was GAU but not when it was GUA. Similar results were found when other non-AUG start sites, the Sendai virus P gene ACG and the c-myc-1 CUG, were examined. These results suggest that positions +5 and +6 are important determinants for initiation at non-AUG start sites, and that they are recognized independently of the overall secondary structure of the mRNA.     

A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent in-frame AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397-1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397-1399 as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that astable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.     

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.     

Mutation and selection on the anticodon of tRNA genes in vertebrate mitochondrial genomes

The H-strand of vertebrate mitochondrial DNA is left single-stranded for hours during the slow DNA replication. This facilitates C-->U mutations on the H-strand (and consequently G-->A mutations on the L-strand) via spontaneous deamination which occurs much more frequently on single-stranded than on double-stranded DNA. For the 12 coding sequences (CDS) collinear with the L-strand, NNY synonymous codon families (where N stands for any of the four nucleotides and Y stands for either C or U) end mostly with C, and NNR and NNN codon families (where R stands for either A or G) end mostly with A. For the lone ND6 gene on the other strand, the codon bias is the opposite, with NNY codon families ending mostly with U and NNR and NNN codon families ending mostly with G. These patterns are consistent with the strand-specific mutation bias. The codon usage biased towards C-ending and A-ending in the 12 CDS sequences affects the codon-anticodon adaptation. The wobble site of the anticodon is always G for NNY codon families dominated by C-ending codons and U for NNR and NNN codon families dominated by A-ending codons. The only, but consistent, exception is the anticodon of tRNA-Met which consistently has a 5'-CAU-3' anticodon base-pairing with the AUG codon (the translation initiation codon) instead of the more frequent AUA. The observed CAU anticodon (matching AUG) would increase the rate of translation initiation but would reduce the rate of peptide elongation because most methionine codons are AUA, whereas the unobserved UAU anticodon (matching AUA) would increase the elongation rate at the cost of translation initiation rate. The consistent CAU anticodon in tRNA-Met suggests the importance of maximizing the rate of translation initiation.     

Analysis of oligonucleotide AUG start codon context in eukariotic mRNAs

The AUG start codon context features have been investigated by analyzing eukaryotic mRNAs belonging to various taxonomic groups. The functional relevance of each specific position surrounding the AUG start codon has been established as a function of the measured shift between base composition observed at that particular position, and base composition averaged over all the 5'untranslated regions. A more detailed analysis carried out on human genes belonging to different isochores showed significant isochore-specific fea-tures that cannot be explained only by a mutational bias effect. The most represented heptamers spanning from position -3 to +4 with respect to the initiator AUG have been determined for mRNAs belonging to different taxonomic groups and a web page utility has been set up (http://bigarea.area.ba.cnr.it:8000/BioWWW/ATG.html) to determine the relative abundance of a user submitted oligonucleotide context in a given species or taxon.     

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λ c I and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.     

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.     

Interrelations between the Nucleotide Context of Human Start AUG Codon,N-end Amino Acids of the Encoded Protein and Initiation of Translation

Abstract

It is known that the recognition of AUG triplet by eukaryotic ribosomes as a translation start site strongly depends on its nucleotide context. However, the relative significance of different context positions is not fully clear. In particular, it concerns the role of 3′-end part of the context located at the beginning of the protein-coding sequence. The significant bias observed in nucleotide frequencies in positions +4, +5, +6 (corresponding to the second codon of CDS) could result from different reasons and their contribution to start codon recognition and initiation of translation is under discussion. In this study, we conducted a comparative computational analysis of the human mRNA samples containing different nucleotides (adenine, guanine or pyrimidine) in the essential context position ?3. It was found that the presence of G in position +4 could be important for the context variant GnnAUG but not for AnnAUG. Interestingly, the second position of proteins encoded by mRNAs with AnnAUG context variant was specifically and significantly enriched with serine whereas the presence of GnnAUG context also correlated with a higher occurrence of alanine and glycine. It is likely that the efficiency of translation initiation process can depend on the interplay between 5′-context part, 3′-context part and the type of amino acid in the second position of the encoded protein.     

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

Background  

The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.     

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.     

Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha

It is by now well established that the estrogen receptor alpha (ER alpha) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5'-untranslated regions (5'-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5'-UTRs of ER alpha contain between one and six upstream open reading frames. In this study the effect of the 5'-UTRs of major human and mouse ER alpha mRNA variants on translation was evaluated. Some of the 5'-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5'-UTRs of ER alpha mRNAs have the potential to regulate estrogen receptor alpha expression.