Specific functional interactions of nucleotides at key <sup>-</sup>3 and <sup>+</sup>4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Pisarev, Andrey V.; Kolupaeva, Victoria G.; Pisareva, Vera P.; Merrick, William C.; Hellen, Christopher U.T.; Pestova, Tatyana V.

doi:10.1101/gad.1397906

Cited by 198 publications

(234 citation statements)

References 36 publications

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“…In particular, eIF1 genes generally contain pyrimidines rather than purines at the Ϫ3 position, shown by Kozak to be the most critical contextual determinant of AUG selection in mammals (24). Using a reconstituted in vitro system, it was demonstrated that mammalian eIF1 can discriminate against AUGs in suboptimal context in addition to preventing recognition of non-AUG codons (35), and it was envisioned that context nucleotides help to stabilize the closed conformation of the PIC that is competent for start codon recognition (37). As first proposed by Ivanov et al (22), its ability to discriminate against poor AUG context would allow mammalian eIF1 to negatively autoregulate translation by discriminating against the poor context of its start codon and, while our work was under way, these researchers provided strong evidence that this autoregulatory mechanism indeed operates in mammalian cells.…”

Section: Metmentioning

confidence: 99%

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Martín-Marcos

Cheung

Hinnebusch

2011

Molecular and Cellular Biology

136

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Yeast eIF1 inhibits initiation at non-AUG triplets, but it was unknown whether it also discriminates against AUGs in suboptimal context. As in other eukaryotes, the yeast gene encoding eIF1 (SUI1) contains an AUG in poor context, which could underlie translational autoregulation. Previously, eIF1 mutations were identified that increase initiation at UUG codons (Sui ؊ phenotype), and we obtained mutations with the opposite phenotype of suppressing UUG initiation (Ssu ؊ phenotype). Remarkably, Sui ؊ mutations in eukaryotic translation initiation factor 1 (eIF1), eIF1A, and eIF2␤ all increase SUI1 expression in a manner diminished by introducing the optimal context at the SUI1 AUG, whereas Ssu ؊ mutations in eIF1 and eIF1A decrease SUI1 expression with the native, but not optimal, context present. Therefore, discrimination against weak context depends on specific residues in eIFs 1, 1A, and 2␤ that also impede selection of non-AUGs, suggesting that context nucleotides and AUG act coordinately to stabilize the preinitiation complex. Although eIF1 autoregulates by discriminating against poor context in yeast and mammals, this mechanism does not prevent eIF1 overproduction in yeast, accounting for the hyperaccuracy phenotype afforded by SUI1 overexpression.Bacterial translation initiation factor 3 (IF3) promotes the fidelity of initiation at AUG codons by discriminating against non-AUG triplets as start sites (17,26,40,45). This discriminatory function forms the basis for IF3's ability to negatively autoregulate translation of its mRNA, which initiates with an AUU start codon (5, 6). IF3 also destabilizes initiation complexes formed on AUG codons at the 5Ј ends of leaderless mRNAs (47), which lack the Shine-Dalgarno sequence that stabilizes mRNA association with the small (30S) ribosomal subunit at the AUG codon.In eukaryotes, the 43S preinitiation complex (PIC), harboring the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and various other eIFs, attaches to the capped 5Ј end of the mRNA and identifies the AUG codon by scanning the mRNA leader base-by-base for complementarity with the anticodon of MettRNA i Met. Efficient initiation is influenced by the sequence immediately upstream from the AUG, but it is unclear how this sequence context is recognized or regulates AUG selection (20,36). The functional counterpart of IF3 in eukaryotes appears to be eIF1 (Sui1 in yeast). eIF1 and IF3 occupy analogous locations on the platform of the small ribosomal subunit (11,27,38) and, similar to IF3, eIF1 blocks formation of stable 48S PICs at near-cognate start codons (20, 36). eIF1/Sui1 and eIF1A (Tif11 in yeast) cooperate to promote an open conformation of the 40S subunit (34) thought to be conducive to scanning (35), and eIF1 also blocks the final step of GTP hydrolysis by the TC, the release of P i from eIF2-GDP-P i , until an AUG enters the P site (1). AUG recognition triggers dissociation of eIF1 from the 40S subunit (30), enabling P i release and stabilizing a closed conformation of the 40S subunit that is incompatible with ...

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Section: Metmentioning

confidence: 99%

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Martín-Marcos

Cheung

Hinnebusch

2011

Molecular and Cellular Biology

136

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“…In brief, positions À3 and þ4 were found to be most important. (1,4) Contexts AnnAUGn and GnnAUGG are considered to be optimal (i.e. they are strong enough to Figure 1.…”

Section: Eukaryotic Mrnas and Translation Initiation Mechanismsmentioning

confidence: 99%

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

2008

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SummaryIt is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.

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“…The release of P i from the 48S complex appears to be coupled with displacement of eIF1 from its binding site near the P site (2,22). In addition, the majority of eIF2 either dissociates from or repositions on the 48S complex following AUG codon recognition (32). Intriguingly, factors eIF3, eIF1, and eIF1A appear to be retained on the 48S complex following eIF2 release (38).…”

mentioning

confidence: 99%

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

Fringer

Acker

Fekete

et al. 2007

Molecular and Cellular Biology

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The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.The initiation of protein synthesis in eukaryotes is a complex series of events orchestrated by initiation factors (eIFs). In vitro studies using purified translation initiation factors have led to the elucidation of the translation initiation pathway in which the different factors associate with the 40S ribosomal subunit and guide the binding of MettRNA i Met and mRNA. While the essential in vitro functions of the different factors are well established, the precise timing of factor binding to and release from the ribosome is less well understood. Moreover, not all of the in vitrodefined functions of the factors have been confirmed in vivo. The first step in translation initiation involves the binding of factor eIF2 to GTP and Met-tRNA i Met forming a ternary complex. The ternary complex along with factors eIF1, eIF1A, eIF3, and eIF5 binds to the 40S ribosome, forming the 43S complex (reviewed in reference 16). The 43S complex then binds to an mRNA near the 5Ј cap, forming a 48S complex that scans along the mRNA in a 3Ј direction in search of an AUG start codon. In the 48S complex, some of the eIF2-GTP is hydrolyzed to eIF2-GDP ϩ P i (2). Upon AUG codon recognition, GTP hydrolysis is completed, and P i is released, converting the 48S complex from an open to a closed complex with Met-tRNA i Met in the P site of the 40S subunit (29). The release of P i from the 48S complex appears to be coupled with displacement of eIF1 from its binding site near the P site (2,22). In addition, the majority of eIF2 either dissociates from or repositions on the 48S complex following AUG codon recognition (32). Intriguingly, factors eIF3, eIF1, and eIF1A appear to be retained on the 48S complex following eIF2 release (38

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Specific functional interactions of nucleotides at key ^-3 and ⁺4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Cited by 198 publications

References 36 publications

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

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Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Cited by 198 publications

References 36 publications

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

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Product

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Specific functional interactions of nucleotides at key ^-3 and ⁺4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex