Regulatory elements in eIF1A control the fielity of start codon selection by modulating tRNA(i) (Met) binding to the ribosome

Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Genes & development (Impact Factor: 10.8). 01/2010; 24(1):97-110. DOI: 10.1101/gad.1871910
Source: PubMed


eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA(i)(Met)) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1) and SE2, was found to stimulate recruitment of Met-tRNA(i)(Met) in the ternary complex (TC) with eIF2.GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1 and SI2, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA(i)(Met) in the P site for AUG selection.

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Available from: Adesh Saini, Feb 25, 2014
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    • "Sui À mutations in the eIF1A SEs destabilize the open/P OUT conformation, allowing transition from the open/P OUT to closed/P IN state at near-cognates, and also reduce the rate of TC loading (Saini et al. 2010), as TC binds most rapidly to the open conformation (Supplemental Fig. S2B; Passmore et al. 2007). Substitution of residues 17–21 in the eIF1A SI element stabilizes the open/P OUT state, which reduces UUG initiation in Sui À mutants—the Ssu À (suppressor of Sui À ) phenotype (Fekete et al. 2007)— and also increases the rate of TC binding (Saini et al. 2010) while decreasing the rate of eIF1 dissociation (Supplemental Fig. S2C; Cheung et al. 2007). tRNA i contains highly conserved sequences not present in elongator tRNAs (Fig. 1A; RajBhandary and Chow 1995; Marck and Grosjean 2002), with important functions in initiation. "
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    ABSTRACT: Eukaryotic initiator tRNA (tRNAi) contains several highly conserved unique sequence features, but their importance in accurate start codon selection was unknown. Here we show that conserved bases throughout tRNAi, from the anticodon stem to acceptor stem, play key roles in ensuring the fidelity of start codon recognition in yeast cells. Substituting the conserved G31:C39 base pair in the anticodon stem with different pairs reduces accuracy (the Sui(-) [suppressor of initiation codon] phenotype), whereas eliminating base pairing increases accuracy (the Ssu(-) [suppressor of Sui(-)] phenotype). The latter defect is fully suppressed by a Sui(-) substitution of T-loop residue A54. These genetic data are paralleled by opposing effects of Sui(-) and Ssu(-) substitutions on the stability of methionylated tRNAi (Met-tRNAi) binding (in the ternary complex [TC] with eIF2-GTP) to reconstituted preinitiation complexes (PICs). Disrupting the C3:G70 base pair in the acceptor stem produces a Sui(-) phenotype and also reduces the rate of TC binding to 40S subunits in vitro and in vivo. Both defects are suppressed by an Ssu(-) substitution in eIF1A that stabilizes the open/POUT conformation of the PIC that exists prior to start codon recognition. Our data indicate that these signature sequences of tRNAi regulate accuracy by distinct mechanisms, promoting the open/POUT conformation of the PIC (for C3:G70) or destabilizing the closed/PIN state (for G31:C39 and A54) that is critical for start codon recognition.
    Preview · Article · Mar 2014 · Genes & development
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    • "Consistent with our model, the Ssu − mutations T15A, D61G, Q84H, and the E48V,L51F double mutation all reduce GCN4-lacZ expression in the SE * mutant strains (Fig. 3C). We previously showed that overexpressing WT eIF1 does not suppress the Gcd − phenotype of this tif11-SE * mutation, even though it stabilizes the open conformation of the PIC (Saini et al. 2010). These data provide evidence for the notion that the eIF1 Ssu − mutations stabilize the P OUT conformation of TC binding (impaired by SE mutations ) in addition to promoting the open conformation of the 40S subunit. "
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    ABSTRACT: In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNAi ternary complex (TC) in a metastable conformation (POUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNAi in the PIN state and preventing completion of GTP hydrolysis (Pi release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu(-) mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui(-)) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu(-) substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the PIN state of TC binding in reconstituted PICs harboring Sui(-) variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the PIN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.
    Preview · Article · Dec 2013 · RNA
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    • "Through the global expression analysis, we have noticed that a eukaryotic translation initiation factor 1A (Eif1a) is highly up-regulated in the Zscan4+ state of ES cells compared with the Zscan4− state.10 Studies using a yeast model have shown that, together with Eif1, Eif1a plays an important role in the identification of translation initiation codon in eukaryotes.11,12 According to the Mouse Genome Informatics database13 at The Jackson Laboratory, Eif1a was originally identified as a mouse eukaryotic elongation factor Tu in erythroleukemic cells.14 "
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    ABSTRACT: Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.
    Full-text · Article · May 2013 · DNA Research
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