Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin.

Recombinant plasmids that direct synthesis of rat preproinsulin under the direction of the SV40 early promoter have been used to probe the mechanism of initiation of translation. Insertion of an upstream AUG triplet that was out-of-frame with respect to the coding sequence for preproinsulin reduced the yield of proinsulin, in keeping with the predictions of the scanning model. The extent to which an upstream AUG codon interfered depended on sequences surrounding the AUG triplet; with two constructs ( p255 /20 and C2) the 5'-proximal AUG codon constituted an absolute barrier: there was no initiation at the downstream start site for preproinsulin. With two other constructs ( p255 /9, p255 /21), however, proinsulin was made despite the presence of an upstream, out-of-frame AUG codon in a favorable context for initiation. In those cases the reading frame set by the first AUG triplet was short, terminating before the start of the preproinsulin coding sequence. The interpretation that ribosomes initiate at the first AUG, terminate, and then reinitiate at the AUG that directly precedes the preproinsulin coding sequence was tested by introducing a point mutation that eliminated the terminator codon: the resulting mutant made no proinsulin.     

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.     

Two Alternative Ways of Start Site Selection in Human Norovirus Reinitiation of Translation

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.     

mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring β-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position ?3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (?3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.     

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.     

Regulation of protein synthesis by mRNA structure

In addition to the m⁷G cap structure, the length of the 5 UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structuresper se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.     

真核细胞中多重表达框mRNA的选择性翻译起始

扫描模型和遗漏扫描模型是真核生物mRNA翻译起始的两种主要机制,但其仍存在某些例外情况,如对具有多顺反子结构的mRNA,选择性翻译起始的发生机制目前仍不清楚.本研究基于GFP蛋白开放表达框(ORF)构建了一系列重组表达载体,用以转录在移码翻译顺序及同一翻译顺序下,AUG起始密码子处于不同序列背景,以及间隔不同距离的多顺反子结构mRNA.通过转染人Bel 7402细胞系,研究了这些多顺反子结构mRNA的翻译起始模式.结果表明,在移码翻译顺序下,多顺反子mRNA可翻译出对应的不同蛋白质,而在同一翻译顺序下,GFP蛋白表达框中的多个AUG密码子,仅有首位起始密码子可发挥作用,提示核糖体在从首位起始密码子开始翻译的同时,可能会有部分核糖体继续向下扫描并识别下游的起始密码子,而这种选择性的翻译起始效率,主要取决于密码子所处的序列背景及间隔距离等因素.     

Control of start codon choice on a plant viral RNA encoding overlapping genes.

The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.     

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

The role of alternative translation start sites in the generation of human protein diversity

According to the scanning model, 40S ribosomal subunits initiate translation at the first (5 proximal) AUG codon they encounter. However, if the first AUG is in a suboptimal context, it may not be recognized, and translation can then initiate at downstream AUG(s). In this way, a single RNA can produce several variant products. Earlier experiments suggested that some of these additional protein variants might be functionally important. We have analysed human mRNAs that have AUG triplets in 5 untranslated regions and mRNAs in which the annotated translational start codon is located in a suboptimal context. It was found that 3% of human mRNAs have the potential to encode N-terminally extended variants of the annotated proteins and 12% could code for N-truncated variants. The predicted subcellular localizations of these protein variants were compared: 31% of the N-extended proteins and 30% of the N-truncated proteins were predicted to localize to subcellular compartments that differed from those targeted by the annotated protein forms. These results suggest that additional AUGs may frequently be exploited for the synthesis of proteins that possess novel functional properties.Electronic Supplementary Material Supplementary material is available for this article at     

Rps3 and rpl16 genes do not overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria?

Characterization of the Oenothera mitochondrial ribosomal gene cluster rps19-rps3-rpl16 shows the two genes rps3 and rpl16 to be separated by 9 nucleotides. The first codon of rpl16 is a GTG codon for valine and the only potential translational start. This GTG codon is conserved at the same position in maize, Petunia and Marchantia mitochondria, while sequences diverge upstream. These observations suggest that GTG at least at this position may act as translation initiation codon in plant mitochondria. Analysis of RNA editing suggests both genes to code for functional ribosomal proteins in Oenothera mitochondria. A duplication/recombination event at a decanucleotide in the intron of rps3 created a pseudogene missing part of the intron and the 3 exon.     

Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.     

The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli

Summary The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the P_L and tac promoters. The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated. The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis. Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon. The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene. In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.     

Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence

The mechanism of translational initiation differs between prokaryotes and eukaryotes. Prokaryotic mRNAs generally contain within their 5′-untranslated region (5′-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site. Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable. Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5′-UTR (−20 to −1 with respect to the start codon). In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation. UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5′-UTR. Insertion of an additional initiation region (SD-sequence and AUG) in the 5′-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet. We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.     

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.     

Interpreting cDNA sequences: Some insights from studies on translation

This review discusses some rules for assessing the completeness of a cDNA sequence and identifying the start site for translation. Features commonly invoked—such as an ATG codon in a favorable context for initiation, or the presence of an upstream in-frame terminator codon, or the prediction of a signal peptide-like sequence at the amino terminus—have some validity; but examples drawn from the literature illustrate limitations to each of these criteria. The best advice is to inspect a cDNA sequence not only for these positive features but also for the absence of certain negative indicators. Three specific warning signs are discussed and documented: (i) The presence of numerous ATG codons upstream from the presumptive start site for translation often indicates an aberration (sometimes a retained intron) at the 5′ end of the cDNA. (ii) Even one strong, upstream, out-of-frame ATG codon poses a problem if the reading frame set by the upstream ATG overlaps the presumptive start of the major open reading frame. Many cDNAs that display this arrangement turn out to be incomplete; that is, the out-of-frame ATG codon is within, rather than upstream from, the protein coding domain. (iii) A very weak context at the putative start site for translation often means that the cDNA lacks the authentic initiator codon. In addition to presenting some criteria that may aid in recognizing incomplete cDNA sequences, the review includes some advice for using in vitro translation systems for the expression of cDNAs. Some unresolved questions about translational regulation are discussed by way of illustrating the importance of verifying mRNA structures before making deductions about translation. Received: 24 April 1996 / Accepted: May 1996