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.     

Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV-irradiation

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.     

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.     

Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.     

真核基因起始与终止密码子旁侧序列特征分析

真核基因起始与终止密码子旁侧序列的特征对于确定cDNA开放阅读框架 (ORF)和预测基因组序列中的编码区 (CDS)非常重要。基于高质量RefSeq数据库 ,在较大数据规模下统计分析了起始密码子旁侧序列所具有的“Kozak规则” ,发现不同物种之间存在差别。同时分析了不同终止密码子旁侧序列的统计学特征 ,给出了相应的正则表达式。由于发现多种基因中存在同相位起始、终止密码子串联使用的情况 ,亦对此进行了讨论。     

Downstream sequence adjacent to AUG affects translation of chloramphenicol acetyl transferase in eukaryotic cells

The CAT gene is widely used as a reporter in eukaryotic systems because of the efficient translation of its mRNA. We report here that a sequence occurring in the CAT mRNA at +15 nucleotides from CAT AUG is essential for translation. This sequence includes a stem-loop structure, which, however, exhibits a calculated stability significantly lower than that required for a hairpin to act as an enhancer of translation in vitro. Replacement of this region with the corresponding sequence from mRNAs that are normally translated in eukaryotic systems drastically reduced translation of CAT in COS cells, although the consensus sequence around the AUG, known to be required for high-level translation initiation, was conserved. These observations may be relevant for the exploitation of the CAT reporter system for analysis of the mechanisms of translation initiation by means of fusion constructs.     

The flatworm spliced leader 3'-terminal AUG as a translation initiator methionine

Spliced leader (SL) RNA trans-splicing contributes the 5' termini to mRNAs in a variety of eukaryotes. In contrast with some transsplicing metazoan groups (e.g. nematodes), flatworm spliced leaders are variable in both sequence and length in different flatworm taxa. However, an absolutely conserved and unique feature of all flatworm spliced leaders is the presence of a 3'-terminal AUG. We previously suggested that the Schistosoma mansoni spliced leader AUG might contribute a required translation initiator methionine to recipient mRNAs. Here we identified and examined trans-spliced cDNAs from a large set of newly available schistosome cDNAs. 28% of the trans-spliced cDNAs have the SL AUG in-frame with the major open reading frame of the mRNA. We identified over 40 cDNAs (40% of the SL AUG in-frame clones) that require the SL AUG as an initiator methionine to synthesize phylogenetically conserved N-terminal residues characteristic of orthologous proteins. RNA transfection experiments using several schistosome stages demonstrated that the flatworm SL AUG can serve as a translation initiator methionine in vivo. We also present in vivo translation studies of the schistosome initiator methionine context and the effect of the spliced leader AUG added upstream and out-of-frame with the main open reading of recipient mRNAs. Overall, our data have provided evidence that another function of flatworm spliced leader trans-splicing is to provide some recipient mRNAs with an initiator methionine for translation initiation.     

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.     

Cooperative effects by the initiation codon and its flanking regions on translation initiation

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.     

Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.     

Structural insights into eukaryotic ribosomes and the initiation of translation

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Highlights? Overview of recent progress in the structural characterization of eukaryotic ribosomes and initiation complexes. ? Crystal structures, cryo-EM and biochemical data are combined to derive structural models of larger assemblies. ? Homology models of eukaryotic initiation complexes provide a starting point for future experiments.     

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.     

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.