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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 Sui− substitution G31R and its intragenic suppressors M18V and G62S on GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. Double mutant derivatives of Sui− G31R eIF5 (G31R,M18V and G31R,G62S) were generated by combining M18V or G62S with G31R in the same recombinant eIF5 variants. The kinetics of Pi formation from the 43S PIC with a model mRNA with an AUG or UUG start codon were measured for these mutants as described in Figure 1B. With WT eIF5, the fast phase of the reaction was shown previously to correspond to GTP hydrolysis and the slow phase to Pi release, which drives GTP hydrolysis to completion (10). (A) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,M18V (triangles) variants of eIF5 with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of G31R,M18V (shown in inset). (B) Observed rate constants for GTP hydrolysis (k1; red and blue striped bars; left Y-axis) and Pi release (k2; red and blue solid bars; right Y-axis) for the cases shown in (A). (C) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,G62S (diamonds) eIF5 variants with AUG (red) or UUG (blue) mRNAs. (D) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the cases shown in (C). (E) GTP hydrolysis and Pi release with WT (circles) or M18V (inverted triangles) eIF5 variants with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of M18V (shown in inset). (F) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the curves shown in (E). Data in (B), (D) and (F) are the averages of three experiments and error bars represent standard errors of the mean.
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Figure 3: Effects of eIF5 Sui− substitution G31R and its intragenic suppressors M18V and G62S on GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. Double mutant derivatives of Sui− G31R eIF5 (G31R,M18V and G31R,G62S) were generated by combining M18V or G62S with G31R in the same recombinant eIF5 variants. The kinetics of Pi formation from the 43S PIC with a model mRNA with an AUG or UUG start codon were measured for these mutants as described in Figure 1B. With WT eIF5, the fast phase of the reaction was shown previously to correspond to GTP hydrolysis and the slow phase to Pi release, which drives GTP hydrolysis to completion (10). (A) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,M18V (triangles) variants of eIF5 with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of G31R,M18V (shown in inset). (B) Observed rate constants for GTP hydrolysis (k1; red and blue striped bars; left Y-axis) and Pi release (k2; red and blue solid bars; right Y-axis) for the cases shown in (A). (C) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,G62S (diamonds) eIF5 variants with AUG (red) or UUG (blue) mRNAs. (D) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the cases shown in (C). (E) GTP hydrolysis and Pi release with WT (circles) or M18V (inverted triangles) eIF5 variants with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of M18V (shown in inset). (F) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the curves shown in (E). Data in (B), (D) and (F) are the averages of three experiments and error bars represent standard errors of the mean.

Mentions: The results above indicate that the M18V substitution in eIF5 confers nearly complete suppression of the Sui− phenotype of G31R, without overcoming the lethality of G31R in cells lacking WT eIF5. Given its location only three residues from a key catalytic residue (Arg-15) in the unstructured NTT, we hypothesized that M18V impairs GAP function in the G31R,M18V double mutant. To test this possibility, we assayed eIF5 stimulation of GTP hydrolysis and Pi release from eIF2 in reconstituted PICs. As described above in Figures 1B and C, the G31R single substitution reduces the rates of GTP hydrolysis and Pi release at AUG while increasing them at UUG, reversing the differential effects of AUG and UUG from those seen with WT eIF5 (Figures 3A and B WT versus G31R and Table 3, rows 1–2). In the G31R,M18V double mutant, M18V reduces the rates of Pi release at both AUG and UUG compared to those seen in the G31R single mutant (Figures 3A and B G31R,M18V versus G31R, k2 values), yielding rate constants at AUG and UUG that are 10- and 20-fold, respectively, below those observed with G31R eIF5 (Table 3, row 4 versus 2; k2 values). M18V in the double mutant also dramatically reduces the rates of GTP hydrolysis at AUG and UUG compared to those seen in the G31R single mutant, by factors of >150-fold (Figures 3A and B G31R,M18V versus G31R and Table 3, row 4 versus 2, k1 values), indicating a major defect in the GAP function of the double mutant. These strong defects in both GTP hydrolysis and Pi release at AUG likely account for the inability of M18V to suppress the lethality of G31R invivo (Figure 2A). Combining M18V with G31R decreases the Pi release rates ∼2-fold more at UUG than at AUG (Figures 3A and B G31R,M18V versus G31R; Table 3, row 4 versus 2, k2 values) to produce a UUG:AUG ratio of Pi release—the rate-limiting step—close to 1 in the double mutant. Although this ratio of release rates is still above that observed with WT eIF5, the trend is consistent with suppression of the Sui− phenotype of G31R by the M18V substitution.


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 Sui− substitution G31R and its intragenic suppressors M18V and G62S on GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. Double mutant derivatives of Sui− G31R eIF5 (G31R,M18V and G31R,G62S) were generated by combining M18V or G62S with G31R in the same recombinant eIF5 variants. The kinetics of Pi formation from the 43S PIC with a model mRNA with an AUG or UUG start codon were measured for these mutants as described in Figure 1B. With WT eIF5, the fast phase of the reaction was shown previously to correspond to GTP hydrolysis and the slow phase to Pi release, which drives GTP hydrolysis to completion (10). (A) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,M18V (triangles) variants of eIF5 with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of G31R,M18V (shown in inset). (B) Observed rate constants for GTP hydrolysis (k1; red and blue striped bars; left Y-axis) and Pi release (k2; red and blue solid bars; right Y-axis) for the cases shown in (A). (C) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,G62S (diamonds) eIF5 variants with AUG (red) or UUG (blue) mRNAs. (D) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the cases shown in (C). (E) GTP hydrolysis and Pi release with WT (circles) or M18V (inverted triangles) eIF5 variants with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of M18V (shown in inset). (F) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the curves shown in (E). Data in (B), (D) and (F) are the averages of three experiments and error bars represent standard errors of the mean.
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Figure 3: Effects of eIF5 Sui− substitution G31R and its intragenic suppressors M18V and G62S on GTP hydrolysis and Pi release from reconstituted 43S·mRNA PICs. Double mutant derivatives of Sui− G31R eIF5 (G31R,M18V and G31R,G62S) were generated by combining M18V or G62S with G31R in the same recombinant eIF5 variants. The kinetics of Pi formation from the 43S PIC with a model mRNA with an AUG or UUG start codon were measured for these mutants as described in Figure 1B. With WT eIF5, the fast phase of the reaction was shown previously to correspond to GTP hydrolysis and the slow phase to Pi release, which drives GTP hydrolysis to completion (10). (A) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,M18V (triangles) variants of eIF5 with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of G31R,M18V (shown in inset). (B) Observed rate constants for GTP hydrolysis (k1; red and blue striped bars; left Y-axis) and Pi release (k2; red and blue solid bars; right Y-axis) for the cases shown in (A). (C) GTP hydrolysis and Pi release with WT (circles), G31R (squares) and G31R,G62S (diamonds) eIF5 variants with AUG (red) or UUG (blue) mRNAs. (D) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the cases shown in (C). (E) GTP hydrolysis and Pi release with WT (circles) or M18V (inverted triangles) eIF5 variants with AUG (red) or UUG (blue) mRNAs. GTP hydrolysis and Pi release was monitored for 4 min in case of M18V (shown in inset). (F) Observed rate constants for GTP hydrolysis (k1) and Pi release (k2) for the curves shown in (E). Data in (B), (D) and (F) are the averages of three experiments and error bars represent standard errors of the mean.
Mentions: The results above indicate that the M18V substitution in eIF5 confers nearly complete suppression of the Sui− phenotype of G31R, without overcoming the lethality of G31R in cells lacking WT eIF5. Given its location only three residues from a key catalytic residue (Arg-15) in the unstructured NTT, we hypothesized that M18V impairs GAP function in the G31R,M18V double mutant. To test this possibility, we assayed eIF5 stimulation of GTP hydrolysis and Pi release from eIF2 in reconstituted PICs. As described above in Figures 1B and C, the G31R single substitution reduces the rates of GTP hydrolysis and Pi release at AUG while increasing them at UUG, reversing the differential effects of AUG and UUG from those seen with WT eIF5 (Figures 3A and B WT versus G31R and Table 3, rows 1–2). In the G31R,M18V double mutant, M18V reduces the rates of Pi release at both AUG and UUG compared to those seen in the G31R single mutant (Figures 3A and B G31R,M18V versus G31R, k2 values), yielding rate constants at AUG and UUG that are 10- and 20-fold, respectively, below those observed with G31R eIF5 (Table 3, row 4 versus 2; k2 values). M18V in the double mutant also dramatically reduces the rates of GTP hydrolysis at AUG and UUG compared to those seen in the G31R single mutant, by factors of >150-fold (Figures 3A and B G31R,M18V versus G31R and Table 3, row 4 versus 2, k1 values), indicating a major defect in the GAP function of the double mutant. These strong defects in both GTP hydrolysis and Pi release at AUG likely account for the inability of M18V to suppress the lethality of G31R invivo (Figure 2A). Combining M18V with G31R decreases the Pi release rates ∼2-fold more at UUG than at AUG (Figures 3A and B G31R,M18V versus G31R; Table 3, row 4 versus 2, k2 values) to produce a UUG:AUG ratio of Pi release—the rate-limiting step—close to 1 in the double mutant. Although this ratio of release rates is still above that observed with WT eIF5, the trend is consistent with suppression of the Sui− phenotype of G31R by the M18V substitution.

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