The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons.

The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast. Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG.     

Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA

TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides.     

AUG is the only initiation codon in eukaryotes

An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia coli and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t6 A hypermodified nucleoside (N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.     

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.     

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.     

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria.     

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.     

Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons

The role which post-translational modification plays in the genesis of herpes simplex virus-induced polypeptides was investigated. Two-dimensional gel electrophoresis was used to identify those polypeptides (i) synthesized in vitro, (ii) labeled in vivo during a pulse, and (iii) labeled after a chase. Excluding glycoproteins, we detected 36 precursor or short-lived polypeptides, 8 polypeptides which were generated by post-translational modification, 46 polypeptides which were apparently not modified after synthesis, and 19 polypeptides which were either transient intermediates or not modified. Comparison of polypeptides synthesized in vitro and during an in vivo pulse showed that translation in vitro resembles quite closely translation in vivo and that amounts of protein synthesized in vivo are determined largely by the levels of mRNA. This analysis provided the basis for an investigation of the suggestion (C.M. Preston and D.J. McGeoch, J. Virol. 38:593-605, 1981) that the two polypeptides of apparent molecular weights of 43,000 (VI 43) and 39,000 (VI 39) encoded by the herpes simplex virus type 1 thymidine kinase gene are translated from a single mRNA by two in-phase initiation codons. Hybrid arrest was used to identify in vitro translation products encoded by the thymidine kinase gene. Two-dimensional gel electrophoresis showed that VI 39 was more acidic than VI 43, consistent with the predicted amino acid composition of a polypeptide whose synthesis was initiated at the second AUG codon, located 135 bases downstream from the first. Furthermore, two-dimensional gels revealed a third polypeptide whose synthesis was arrested by the same fragment. Its pI and apparent molecular weight (38,000) were compatible with initiation of translation at a third AUG codon an additional 42 bases downstream. Our findings provide strong evidence that downstream initiation codons within the thymidine kinase mRNA are used.     

Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism.

The phosphoprotein of rabies virus is a 297-amino-acid polypeptide encoded by the longest open reading frame of the P gene. Immunoprecipitation experiments using a monoclonal antiserum directed against the P protein detected the P protein and at least four additional shorter products in infected cells, cells transfected with a plasmid encoding the wild-type P protein, and purified virus (CVS strain). By means of deletion analyses, these proteins were shown to be translated from secondary downstream in-frame AUG initiation codons. Immunofluorescence experiments indicated that all these P products were found in the cytoplasm of transfected cells; however, the proteins initiated from the third, fourth, and fifth AUG codons were found mostly in the nucleus. Changes in the 5'-terminal region of the P mRNA (including site-specific mutations, deletions, and insertions) demonstrated that a leaky scanning mechanism is responsible for translation initiation of the P gene at several sites.     

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.