Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins

Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex–CAT or Tax–CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex–CAT and Tax–CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.     

Translation initiation by non-AUG codons in Arabidopsis thaliana transgenic plants

The efficiency of translation initiation at codons differing at one or two nucleotides from AUG was tested as initiation codons for the phosphinotricin-acetyltransferase gene in T-DNA plant transformation in Arabidopsis thaliana. With the exception of UUA codon that differs from AUG at two nucleotides and does not permit any detectable activity, all the other codons (AUC, GUG, ACG, and CUG) present a phosphinotrycin acetyltransferase activity that varies between 5 and 10% of the AUG activity. This low activity is sufficient to confer glufosinate resistance to some of the plants. These results indicate that, in plants as is the case in animals, non-AUG initiating codons may be used for translation initiation, namely when a low expression rate is needed.     

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.     

Principles of start codon recognition in eukaryotic translation initiation

Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit.     

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.     

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.     

Effect of structure of the initiator codon on translation in E. coli

A set of plasmids carrying different initiator codons--either AUG, or GUG, or UUG, or CUG (as a control) in the hybrid gene lacIZ--was constructed by using synthetic oligonucleotides. GUG and UUG codons were demonstrated to be 2-3 times less effective than AUG in translation initiation. Furthermore, the correlation between the efficiencies of different initiator codons in translation initiation proved to vary, depending on the phase of bacterial growth. The rarely occurring usage in nature of the initiator codons GUG and UUG is supposed to be due to the particular role played by the initiator triplets in regulation of gene expression.     

Truncated forms of the dual function human ASCT2 neutral amino acid transporter/retroviral receptor are translationally initiated at multiple alternative CUG and GUG codons

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.     

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.     

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.     

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.     

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.     

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.     

The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5' untranslated leader

Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless cI mRNA from bacteriophage lambda. In a cI-lacZ translational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels. Addition of an untranslated lac leader and Shine-Dalgarno sequence to cI increased expression but still showed a dependence on an AUG for maximum expression. cI-lacZ mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5' untranslated leader.