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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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Effects of eIF5 intragenic suppressors M18V and G62S on the kinetics of eIF1A dissociation from reconstituted 43S·mRNA PICs. Dissociation of fluorescein-labeled eIF1A from 43S·mRNA complexes assembled with WT or mutant eIF5 was monitored by following the change in fluorescence anisotropy over time after the addition of a large excess of unlabeled WT eIF1A. The data were fit with a double exponential decay equation. Previous data indicate that the fast phase corresponds to dissociation of eIF1A from the ‘open’ conformation of the PIC and the second phase corresponds to dissociation from the ‘closed’ state. In order to highlight differences in the fast phases, the early time points of the respective eIF1A dissociation curves are shown in insets. The ratio of the amplitudes of the second phase (closed state) to first phase (open state) is defined as Kamp. (A) eIF1A dissociation from PICs assembled with WT (circles) and G31R (squares) eIF5 with mRNA(AUG) (red; Kamp for WT is 6.0 ± 0.7 and for G31R is 3.0 ± 0.2) and mRNA(UUG) (blue; Kamp for WT is 4.0 ± 0.2 and for G31R is 10 ± 0.6). (B) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,M18V (triangles) eIF5 with mRNA(AUG) (red; Kamp is 2.0 ± 0.1 for G31R,M18V) and mRNA(UUG) (blue; Kamp is 9.0 ± 1.0 for G31R,M18V)) mRNA. (C) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 1.1 ± 0.04 for G31R,G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.3 for G31R,G62S). (D) eIF1A dissociation from PICs assembled with WT (circles) and G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 3.0 ± 0.05 for G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.06 for G62S). All experiments were performed at least three times and errors are standard errors of the mean.
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Figure 4: Effects of eIF5 intragenic suppressors M18V and G62S on the kinetics of eIF1A dissociation from reconstituted 43S·mRNA PICs. Dissociation of fluorescein-labeled eIF1A from 43S·mRNA complexes assembled with WT or mutant eIF5 was monitored by following the change in fluorescence anisotropy over time after the addition of a large excess of unlabeled WT eIF1A. The data were fit with a double exponential decay equation. Previous data indicate that the fast phase corresponds to dissociation of eIF1A from the ‘open’ conformation of the PIC and the second phase corresponds to dissociation from the ‘closed’ state. In order to highlight differences in the fast phases, the early time points of the respective eIF1A dissociation curves are shown in insets. The ratio of the amplitudes of the second phase (closed state) to first phase (open state) is defined as Kamp. (A) eIF1A dissociation from PICs assembled with WT (circles) and G31R (squares) eIF5 with mRNA(AUG) (red; Kamp for WT is 6.0 ± 0.7 and for G31R is 3.0 ± 0.2) and mRNA(UUG) (blue; Kamp for WT is 4.0 ± 0.2 and for G31R is 10 ± 0.6). (B) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,M18V (triangles) eIF5 with mRNA(AUG) (red; Kamp is 2.0 ± 0.1 for G31R,M18V) and mRNA(UUG) (blue; Kamp is 9.0 ± 1.0 for G31R,M18V)) mRNA. (C) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 1.1 ± 0.04 for G31R,G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.3 for G31R,G62S). (D) eIF1A dissociation from PICs assembled with WT (circles) and G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 3.0 ± 0.05 for G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.06 for G62S). All experiments were performed at least three times and errors are standard errors of the mean.

Mentions: 43S·mRNA(AUG) or 43S·mRNA(UUG) complexes were assembled with eIF1A (labeled at its C-terminus with fluorescein) in the presence of eIF5, chased with excess unlabeled eIF1A, and eIF1A dissociation was measured over time as the decrease in fluorescence anisotropy. Dissociation of WT eIF1A from the PIC is biphasic, with rate constants for the fast and slow phases designated k1 and k2, respectively. We defined an apparent equilibrium constant, Kamp, as the ratio of amplitudes of the slow to fast phases (α2/α1). Accordingly, Kamp values >1 indicate that the slow phase dominates the reaction. In accordance with previous findings (12), WT eIF1A dissociates more slowly from AUG versus UUG complexes (Figure 4A), as dissociation from the AUG complexes is dominated by the slow phase (Kamp of 6.0; Table 4, row 1), while replacing AUG with UUG reduces Kamp to 4.0 and also increases k2 by ∼3-fold (row 2). These findings suggest that only approximately one-seventh of the PICs are in the open conformation at AUG (Kamp of 6.0) whereas approximately one-fifth occupy the open conformation at UUG (Kamp of 4.0), and the ∼3-fold higher value of k2 at UUG indicates that eIF1A is bound less stably in the closed conformation at UUG versus AUG. This latter finding could reflect a decreased strength of interaction between eIF1A and eIF5, which we have shown is a key event in mediating a proper response to start codon recognition (3,12).


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)

Effects of eIF5 intragenic suppressors M18V and G62S on the kinetics of eIF1A dissociation from reconstituted 43S·mRNA PICs. Dissociation of fluorescein-labeled eIF1A from 43S·mRNA complexes assembled with WT or mutant eIF5 was monitored by following the change in fluorescence anisotropy over time after the addition of a large excess of unlabeled WT eIF1A. The data were fit with a double exponential decay equation. Previous data indicate that the fast phase corresponds to dissociation of eIF1A from the ‘open’ conformation of the PIC and the second phase corresponds to dissociation from the ‘closed’ state. In order to highlight differences in the fast phases, the early time points of the respective eIF1A dissociation curves are shown in insets. The ratio of the amplitudes of the second phase (closed state) to first phase (open state) is defined as Kamp. (A) eIF1A dissociation from PICs assembled with WT (circles) and G31R (squares) eIF5 with mRNA(AUG) (red; Kamp for WT is 6.0 ± 0.7 and for G31R is 3.0 ± 0.2) and mRNA(UUG) (blue; Kamp for WT is 4.0 ± 0.2 and for G31R is 10 ± 0.6). (B) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,M18V (triangles) eIF5 with mRNA(AUG) (red; Kamp is 2.0 ± 0.1 for G31R,M18V) and mRNA(UUG) (blue; Kamp is 9.0 ± 1.0 for G31R,M18V)) mRNA. (C) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 1.1 ± 0.04 for G31R,G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.3 for G31R,G62S). (D) eIF1A dissociation from PICs assembled with WT (circles) and G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 3.0 ± 0.05 for G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.06 for G62S). All experiments were performed at least three times and errors are standard errors of the mean.
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Figure 4: Effects of eIF5 intragenic suppressors M18V and G62S on the kinetics of eIF1A dissociation from reconstituted 43S·mRNA PICs. Dissociation of fluorescein-labeled eIF1A from 43S·mRNA complexes assembled with WT or mutant eIF5 was monitored by following the change in fluorescence anisotropy over time after the addition of a large excess of unlabeled WT eIF1A. The data were fit with a double exponential decay equation. Previous data indicate that the fast phase corresponds to dissociation of eIF1A from the ‘open’ conformation of the PIC and the second phase corresponds to dissociation from the ‘closed’ state. In order to highlight differences in the fast phases, the early time points of the respective eIF1A dissociation curves are shown in insets. The ratio of the amplitudes of the second phase (closed state) to first phase (open state) is defined as Kamp. (A) eIF1A dissociation from PICs assembled with WT (circles) and G31R (squares) eIF5 with mRNA(AUG) (red; Kamp for WT is 6.0 ± 0.7 and for G31R is 3.0 ± 0.2) and mRNA(UUG) (blue; Kamp for WT is 4.0 ± 0.2 and for G31R is 10 ± 0.6). (B) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,M18V (triangles) eIF5 with mRNA(AUG) (red; Kamp is 2.0 ± 0.1 for G31R,M18V) and mRNA(UUG) (blue; Kamp is 9.0 ± 1.0 for G31R,M18V)) mRNA. (C) eIF1A dissociation from PICs assembled with G31R (squares) and G31R,G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 1.1 ± 0.04 for G31R,G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.3 for G31R,G62S). (D) eIF1A dissociation from PICs assembled with WT (circles) and G62S (diamonds) eIF5 with mRNA(AUG) (red; Kamp is 3.0 ± 0.05 for G62S) and mRNA(UUG) (blue; Kamp is 2.3 ± 0.06 for G62S). All experiments were performed at least three times and errors are standard errors of the mean.
Mentions: 43S·mRNA(AUG) or 43S·mRNA(UUG) complexes were assembled with eIF1A (labeled at its C-terminus with fluorescein) in the presence of eIF5, chased with excess unlabeled eIF1A, and eIF1A dissociation was measured over time as the decrease in fluorescence anisotropy. Dissociation of WT eIF1A from the PIC is biphasic, with rate constants for the fast and slow phases designated k1 and k2, respectively. We defined an apparent equilibrium constant, Kamp, as the ratio of amplitudes of the slow to fast phases (α2/α1). Accordingly, Kamp values >1 indicate that the slow phase dominates the reaction. In accordance with previous findings (12), WT eIF1A dissociates more slowly from AUG versus UUG complexes (Figure 4A), as dissociation from the AUG complexes is dominated by the slow phase (Kamp of 6.0; Table 4, row 1), while replacing AUG with UUG reduces Kamp to 4.0 and also increases k2 by ∼3-fold (row 2). These findings suggest that only approximately one-seventh of the PICs are in the open conformation at AUG (Kamp of 6.0) whereas approximately one-fifth occupy the open conformation at UUG (Kamp of 4.0), and the ∼3-fold higher value of k2 at UUG indicates that eIF1A is bound less stably in the closed conformation at UUG versus AUG. This latter finding could reflect a decreased strength of interaction between eIF1A and eIF5, which we have shown is a key event in mediating a proper response to start codon recognition (3,12).

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