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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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TIF5 suppressors confer allele-specific suppression of Sui− mutations in eIF2β, eIF1A and eIF1. (A) Slg− and His+/Sui− phenotypes of derivatives of his4-301 strain ASY100 harboring the indicated TIF5 alleles on LEU2 plasmid and either YCpSUI3-2 plasmid harboring SUI3-2 (rows 2–7) or empty vector (row 1) were determined by spotting serial 10-fold dilutions on SC medium lacking leucine and tryptophan (SC-LW) supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (B) Strains described in (A) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in Figure 2C. (C) Derivatives of his4-301 tif11Δ PGAL-TIF5 strain PMY17 harboring a TRP1 plasmid with WT TIF11 (pAS5-142) (lane 1) or tif11-SE1*,SE2*+F131 (pAS5-130) (Lanes 2–7) were transformed with LEU2 plasmids containing the indicated TIF5 alleles, and Slg− and His+/Sui− phenotypes were determined as in (A). (D) Strains described in (C) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in (B). (E) Dominant His−/Ssu− and Slg+ phenotypes of the strains in (C) were analyzed by spotting serial 10-fold dilutions on SC medium containing galactose and lacking tryptophan and leucine supplemented with 0.3 mM His (Gal + His) or 0.0003 mM His (Gal −His) and incubating for 3d (Gal + His) or 6d (Gal − His) at 30°C. (F) Slg− and His+/Sui− phenotypes were determined for derivatives of his4-301 sui1Δ PGAL-TIF5 strain PMY01 harboring sui1-93-97 on a TRP1 plasmid and LEU2 plasmids containing the indicated TIF5 alleles were determined as in (C). (G) Strains described in (F) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as described in (D). For panels A, C and F, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
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Figure 5: TIF5 suppressors confer allele-specific suppression of Sui− mutations in eIF2β, eIF1A and eIF1. (A) Slg− and His+/Sui− phenotypes of derivatives of his4-301 strain ASY100 harboring the indicated TIF5 alleles on LEU2 plasmid and either YCpSUI3-2 plasmid harboring SUI3-2 (rows 2–7) or empty vector (row 1) were determined by spotting serial 10-fold dilutions on SC medium lacking leucine and tryptophan (SC-LW) supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (B) Strains described in (A) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in Figure 2C. (C) Derivatives of his4-301 tif11Δ PGAL-TIF5 strain PMY17 harboring a TRP1 plasmid with WT TIF11 (pAS5-142) (lane 1) or tif11-SE1*,SE2*+F131 (pAS5-130) (Lanes 2–7) were transformed with LEU2 plasmids containing the indicated TIF5 alleles, and Slg− and His+/Sui− phenotypes were determined as in (A). (D) Strains described in (C) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in (B). (E) Dominant His−/Ssu− and Slg+ phenotypes of the strains in (C) were analyzed by spotting serial 10-fold dilutions on SC medium containing galactose and lacking tryptophan and leucine supplemented with 0.3 mM His (Gal + His) or 0.0003 mM His (Gal −His) and incubating for 3d (Gal + His) or 6d (Gal − His) at 30°C. (F) Slg− and His+/Sui− phenotypes were determined for derivatives of his4-301 sui1Δ PGAL-TIF5 strain PMY01 harboring sui1-93-97 on a TRP1 plasmid and LEU2 plasmids containing the indicated TIF5 alleles were determined as in (C). (G) Strains described in (F) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as described in (D). For panels A, C and F, images have been cropped from results obtained from different plates examined in parallel in the same experiments.

Mentions: We sought next to obtain genetic evidence that the Ssu− phenotypes of the M18V and G62S eIF5 substitutions involve distinct molecular mechanisms invivo. To this end, we asked whether M18V and G62S differ significantly in their ability to suppress Sui− substitutions in other initiation factors, beginning with the dominant Sui− mutation SUI3–2, encoding the S264Y substitution in eIF2β. Plasmid-borne SUI3–2 was introduced into SUI3+ tif5Δ strains also containing TIF5 alleles harboring single suppressor mutations (i.e. without G31R). As expected, in the TIF5+ his4–303 strain, SUI3–2 confers growth on −His medium (Figure 5A, rows 1–2) and elevates the UUG:AUG ratio above ∼0.3 (Figure 5B, columns1–2). The His+/Sui− phenotype of SUI3–2 is suppressed efficiently by the TIF5-M18V and TIF5-K33E alleles (Figure 5A, rows 2–4), with commensurate strong reductions in the UUG:AUG initiation ratio (Figure 5B, columns 2–4). Thus, these two eIF5 mutants are effective suppressors of the Sui− phenotypes of both SUI3–2 and (as shown above) the G31R allele of TIF5. Similarly, the L61A mutation, an ineffective suppressor of G31R/SUI5 (Figure 2) likewise only partially suppresses the His+/Sui− phenotype of SUI3–2 (Figure 5A, row 6 versus 2) and is relatively ineffective in reducing the elevated UUG:AUG ratio conferred by SUI3–2 (Figure 5B, columns 6 versus 2). It is interesting however that G62S, one of the strongest suppressors of G31R (Figure 2C and E), is a relatively inefficient suppressor of SUI3–2, failing to eliminate the His+ phenotype (Figure 5A, row 5) and reducing the UUG:AUG ratio by less than a factor of 2 (Figure 5B, column 6 versus 2). This last finding suggests that the Sui− phenotypes of tif5-G31R and SUI3–2 involve at least partially distinct mechanisms and that the G62S substitution in eIF5 more effectively compensates for the defect(s) conferred by tif5-G31R versus SUI3–2.


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)

TIF5 suppressors confer allele-specific suppression of Sui− mutations in eIF2β, eIF1A and eIF1. (A) Slg− and His+/Sui− phenotypes of derivatives of his4-301 strain ASY100 harboring the indicated TIF5 alleles on LEU2 plasmid and either YCpSUI3-2 plasmid harboring SUI3-2 (rows 2–7) or empty vector (row 1) were determined by spotting serial 10-fold dilutions on SC medium lacking leucine and tryptophan (SC-LW) supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (B) Strains described in (A) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in Figure 2C. (C) Derivatives of his4-301 tif11Δ PGAL-TIF5 strain PMY17 harboring a TRP1 plasmid with WT TIF11 (pAS5-142) (lane 1) or tif11-SE1*,SE2*+F131 (pAS5-130) (Lanes 2–7) were transformed with LEU2 plasmids containing the indicated TIF5 alleles, and Slg− and His+/Sui− phenotypes were determined as in (A). (D) Strains described in (C) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in (B). (E) Dominant His−/Ssu− and Slg+ phenotypes of the strains in (C) were analyzed by spotting serial 10-fold dilutions on SC medium containing galactose and lacking tryptophan and leucine supplemented with 0.3 mM His (Gal + His) or 0.0003 mM His (Gal −His) and incubating for 3d (Gal + His) or 6d (Gal − His) at 30°C. (F) Slg− and His+/Sui− phenotypes were determined for derivatives of his4-301 sui1Δ PGAL-TIF5 strain PMY01 harboring sui1-93-97 on a TRP1 plasmid and LEU2 plasmids containing the indicated TIF5 alleles were determined as in (C). (G) Strains described in (F) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as described in (D). For panels A, C and F, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
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Figure 5: TIF5 suppressors confer allele-specific suppression of Sui− mutations in eIF2β, eIF1A and eIF1. (A) Slg− and His+/Sui− phenotypes of derivatives of his4-301 strain ASY100 harboring the indicated TIF5 alleles on LEU2 plasmid and either YCpSUI3-2 plasmid harboring SUI3-2 (rows 2–7) or empty vector (row 1) were determined by spotting serial 10-fold dilutions on SC medium lacking leucine and tryptophan (SC-LW) supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (B) Strains described in (A) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in Figure 2C. (C) Derivatives of his4-301 tif11Δ PGAL-TIF5 strain PMY17 harboring a TRP1 plasmid with WT TIF11 (pAS5-142) (lane 1) or tif11-SE1*,SE2*+F131 (pAS5-130) (Lanes 2–7) were transformed with LEU2 plasmids containing the indicated TIF5 alleles, and Slg− and His+/Sui− phenotypes were determined as in (A). (D) Strains described in (C) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as in (B). (E) Dominant His−/Ssu− and Slg+ phenotypes of the strains in (C) were analyzed by spotting serial 10-fold dilutions on SC medium containing galactose and lacking tryptophan and leucine supplemented with 0.3 mM His (Gal + His) or 0.0003 mM His (Gal −His) and incubating for 3d (Gal + His) or 6d (Gal − His) at 30°C. (F) Slg− and His+/Sui− phenotypes were determined for derivatives of his4-301 sui1Δ PGAL-TIF5 strain PMY01 harboring sui1-93-97 on a TRP1 plasmid and LEU2 plasmids containing the indicated TIF5 alleles were determined as in (C). (G) Strains described in (F) harboring the AUG or UUG HIS4-lacZ reporters were analyzed as described in (D). For panels A, C and F, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
Mentions: We sought next to obtain genetic evidence that the Ssu− phenotypes of the M18V and G62S eIF5 substitutions involve distinct molecular mechanisms invivo. To this end, we asked whether M18V and G62S differ significantly in their ability to suppress Sui− substitutions in other initiation factors, beginning with the dominant Sui− mutation SUI3–2, encoding the S264Y substitution in eIF2β. Plasmid-borne SUI3–2 was introduced into SUI3+ tif5Δ strains also containing TIF5 alleles harboring single suppressor mutations (i.e. without G31R). As expected, in the TIF5+ his4–303 strain, SUI3–2 confers growth on −His medium (Figure 5A, rows 1–2) and elevates the UUG:AUG ratio above ∼0.3 (Figure 5B, columns1–2). The His+/Sui− phenotype of SUI3–2 is suppressed efficiently by the TIF5-M18V and TIF5-K33E alleles (Figure 5A, rows 2–4), with commensurate strong reductions in the UUG:AUG initiation ratio (Figure 5B, columns 2–4). Thus, these two eIF5 mutants are effective suppressors of the Sui− phenotypes of both SUI3–2 and (as shown above) the G31R allele of TIF5. Similarly, the L61A mutation, an ineffective suppressor of G31R/SUI5 (Figure 2) likewise only partially suppresses the His+/Sui− phenotype of SUI3–2 (Figure 5A, row 6 versus 2) and is relatively ineffective in reducing the elevated UUG:AUG ratio conferred by SUI3–2 (Figure 5B, columns 6 versus 2). It is interesting however that G62S, one of the strongest suppressors of G31R (Figure 2C and E), is a relatively inefficient suppressor of SUI3–2, failing to eliminate the His+ phenotype (Figure 5A, row 5) and reducing the UUG:AUG ratio by less than a factor of 2 (Figure 5B, column 6 versus 2). This last finding suggests that the Sui− phenotypes of tif5-G31R and SUI3–2 involve at least partially distinct mechanisms and that the G62S substitution in eIF5 more effectively compensates for the defect(s) conferred by tif5-G31R versus SUI3–2.

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