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Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

Saini AK, Nanda JS, Martin-Marcos P, Dong J, Zhang F, Bhardwaj M, Lorsch JR, Hinnebusch AG - Nucleic Acids Res. (2014)

Bottom Line: Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation.The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors.We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

View Article: PubMed Central - PubMed

Affiliation: 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 Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA sainiade@gmail.com.

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(A) Model describing conformational rearrangements of the PIC during scanning and start codon recognition. (i) eIF1 and the SE elements in the eIF1A CTT stabilize an open 40S conformation to which TC loads rapidly. (ii) The 43S PIC in the open conformation scans for an AUG codon with Met-tRNAi in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis to produce GDP·Pi, but Pi release is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1·40S association. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1. Movement of eIF1 disrupts its interaction with the eIF2β-NTT, which interacts with the eIF5-CTD instead. eIF1 dissociates from the 40S subunit, and the eIF1A SE elements interact with the eIF5-NTD to facilitate Pi release. (Below) Arrows summarize that eIF1 and eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from (3,29)). (B) eIF5 Sui− substitution G31R alters start codon regulation of GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. The kinetics of GTP hydrolysis and Pi release from 43S PICs was measured after addition of WT eIF5 or G31R eIF5 and mRNAs (AUG or UUG). Aliquots from the reactions were quenched at different times with 100 mM EDTA. γ-32P-GTP and γ-32Pi were then separated using PEI-cellulose TLC and quantified by phosphorimager analysis. The fraction of GTP hydrolyzed versus time was plotted and the data fit with a double exponential rate equation. The fast phase corresponds to GTP hydrolysis and the slower phase to Pi release (10). The curves shown are WT eIF5 and AUG mRNA (red circles); WT eIF5 and UUG mRNA (blue circles); G31R eIF5 and AUG mRNA (red squares); or G31R eIF5 and UUG mRNA (blue squares). (C) Histograms showing the observed rate constants for GTP hydrolysis (k1; left Y-axis) and Pi release (k2; right Y-axis) from (B).
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Figure 1: (A) Model describing conformational rearrangements of the PIC during scanning and start codon recognition. (i) eIF1 and the SE elements in the eIF1A CTT stabilize an open 40S conformation to which TC loads rapidly. (ii) The 43S PIC in the open conformation scans for an AUG codon with Met-tRNAi in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis to produce GDP·Pi, but Pi release is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1·40S association. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1. Movement of eIF1 disrupts its interaction with the eIF2β-NTT, which interacts with the eIF5-CTD instead. eIF1 dissociates from the 40S subunit, and the eIF1A SE elements interact with the eIF5-NTD to facilitate Pi release. (Below) Arrows summarize that eIF1 and eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from (3,29)). (B) eIF5 Sui− substitution G31R alters start codon regulation of GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. The kinetics of GTP hydrolysis and Pi release from 43S PICs was measured after addition of WT eIF5 or G31R eIF5 and mRNAs (AUG or UUG). Aliquots from the reactions were quenched at different times with 100 mM EDTA. γ-32P-GTP and γ-32Pi were then separated using PEI-cellulose TLC and quantified by phosphorimager analysis. The fraction of GTP hydrolyzed versus time was plotted and the data fit with a double exponential rate equation. The fast phase corresponds to GTP hydrolysis and the slower phase to Pi release (10). The curves shown are WT eIF5 and AUG mRNA (red circles); WT eIF5 and UUG mRNA (blue circles); G31R eIF5 and AUG mRNA (red squares); or G31R eIF5 and UUG mRNA (blue squares). (C) Histograms showing the observed rate constants for GTP hydrolysis (k1; left Y-axis) and Pi release (k2; right Y-axis) from (B).

Mentions: In translation initiation by the scanning mechanism, the small (40S) ribosomal subunit harboring initiator methionyl tRNA (Met-tRNAi) bound to eIF2-GTP in a ternary complex (TC) attaches near the capped 5′ end of the mRNA and scans the leader for an AUG triplet in optimal sequence context (reviewed in (1,2)). According to our current model (Figure 1A), eIF1 and eIF1A promote an ‘open’ conformation of the 40S subunit that is competent for binding the TC in a metastable state (POUT) that allows the Met-tRNAi to sample successive triplets entering the P site for complementarity to the anticodon triplet. The GTPase activating protein (GAP) eIF5 stimulates Guanosine-5'-triphosphate (GTP) hydrolysis by the TC, but completion of the reaction with release of Pi is blocked by eIF1 in the scanning preinitiation complex (PIC). Base-pairing of Met-tRNAi with an AUG triplet evokes a rearrangement of factors in the PIC, including displacement of eIF1 and the C-terminal tail (CTT) of eIF1A from their locations near the P site, and movement of the eIF1A CTT toward the GAP domain of eIF5. These rearrangements enable dissociation of eIF1 from the 40S subunit, evoking a closed, scanning-arrested conformation of the 40S subunit, Pi release from eIF2-GDP, and tighter binding of Met-tRNAi in the P site (PIN state) (reviewed in (2,3)).


Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

Saini AK, Nanda JS, Martin-Marcos P, Dong J, Zhang F, Bhardwaj M, Lorsch JR, Hinnebusch AG - Nucleic Acids Res. (2014)

(A) Model describing conformational rearrangements of the PIC during scanning and start codon recognition. (i) eIF1 and the SE elements in the eIF1A CTT stabilize an open 40S conformation to which TC loads rapidly. (ii) The 43S PIC in the open conformation scans for an AUG codon with Met-tRNAi in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis to produce GDP·Pi, but Pi release is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1·40S association. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1. Movement of eIF1 disrupts its interaction with the eIF2β-NTT, which interacts with the eIF5-CTD instead. eIF1 dissociates from the 40S subunit, and the eIF1A SE elements interact with the eIF5-NTD to facilitate Pi release. (Below) Arrows summarize that eIF1 and eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from (3,29)). (B) eIF5 Sui− substitution G31R alters start codon regulation of GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. The kinetics of GTP hydrolysis and Pi release from 43S PICs was measured after addition of WT eIF5 or G31R eIF5 and mRNAs (AUG or UUG). Aliquots from the reactions were quenched at different times with 100 mM EDTA. γ-32P-GTP and γ-32Pi were then separated using PEI-cellulose TLC and quantified by phosphorimager analysis. The fraction of GTP hydrolyzed versus time was plotted and the data fit with a double exponential rate equation. The fast phase corresponds to GTP hydrolysis and the slower phase to Pi release (10). The curves shown are WT eIF5 and AUG mRNA (red circles); WT eIF5 and UUG mRNA (blue circles); G31R eIF5 and AUG mRNA (red squares); or G31R eIF5 and UUG mRNA (blue squares). (C) Histograms showing the observed rate constants for GTP hydrolysis (k1; left Y-axis) and Pi release (k2; right Y-axis) from (B).
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Figure 1: (A) Model describing conformational rearrangements of the PIC during scanning and start codon recognition. (i) eIF1 and the SE elements in the eIF1A CTT stabilize an open 40S conformation to which TC loads rapidly. (ii) The 43S PIC in the open conformation scans for an AUG codon with Met-tRNAi in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis to produce GDP·Pi, but Pi release is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1·40S association. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1. Movement of eIF1 disrupts its interaction with the eIF2β-NTT, which interacts with the eIF5-CTD instead. eIF1 dissociates from the 40S subunit, and the eIF1A SE elements interact with the eIF5-NTD to facilitate Pi release. (Below) Arrows summarize that eIF1 and eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from (3,29)). (B) eIF5 Sui− substitution G31R alters start codon regulation of GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. The kinetics of GTP hydrolysis and Pi release from 43S PICs was measured after addition of WT eIF5 or G31R eIF5 and mRNAs (AUG or UUG). Aliquots from the reactions were quenched at different times with 100 mM EDTA. γ-32P-GTP and γ-32Pi were then separated using PEI-cellulose TLC and quantified by phosphorimager analysis. The fraction of GTP hydrolyzed versus time was plotted and the data fit with a double exponential rate equation. The fast phase corresponds to GTP hydrolysis and the slower phase to Pi release (10). The curves shown are WT eIF5 and AUG mRNA (red circles); WT eIF5 and UUG mRNA (blue circles); G31R eIF5 and AUG mRNA (red squares); or G31R eIF5 and UUG mRNA (blue squares). (C) Histograms showing the observed rate constants for GTP hydrolysis (k1; left Y-axis) and Pi release (k2; right Y-axis) from (B).
Mentions: In translation initiation by the scanning mechanism, the small (40S) ribosomal subunit harboring initiator methionyl tRNA (Met-tRNAi) bound to eIF2-GTP in a ternary complex (TC) attaches near the capped 5′ end of the mRNA and scans the leader for an AUG triplet in optimal sequence context (reviewed in (1,2)). According to our current model (Figure 1A), eIF1 and eIF1A promote an ‘open’ conformation of the 40S subunit that is competent for binding the TC in a metastable state (POUT) that allows the Met-tRNAi to sample successive triplets entering the P site for complementarity to the anticodon triplet. The GTPase activating protein (GAP) eIF5 stimulates Guanosine-5'-triphosphate (GTP) hydrolysis by the TC, but completion of the reaction with release of Pi is blocked by eIF1 in the scanning preinitiation complex (PIC). Base-pairing of Met-tRNAi with an AUG triplet evokes a rearrangement of factors in the PIC, including displacement of eIF1 and the C-terminal tail (CTT) of eIF1A from their locations near the P site, and movement of the eIF1A CTT toward the GAP domain of eIF5. These rearrangements enable dissociation of eIF1 from the 40S subunit, evoking a closed, scanning-arrested conformation of the 40S subunit, Pi release from eIF2-GDP, and tighter binding of Met-tRNAi in the P site (PIN state) (reviewed in (2,3)).

Bottom Line: Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation.The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors.We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

View Article: PubMed Central - PubMed

Affiliation: 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 Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA sainiade@gmail.com.

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Related in: MedlinePlus