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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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Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
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Figure 2: Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.

Mentions: M18V strongly suppresses the dominant His+/Sui− phenotype, but not the recessive lethality, conferred by G31R. Thus, transformants harboring tif5-G31R,M18V fail to grow on −His medium in the presence of TIF5+, indicating the Ssu− phenotype, and also cannot grow on 5-FOA medium, indicating inviability of the cells following eviction of TIF5+ (Figure 2A, rows 3–4). The failure to grow on the −His medium does not result from a general growth defect, as the tif5-G31R,M18V strain grows better on +His medium than does the parental His+tif5-G31R strain, which displays the dominant Slg− phenotype conferred by G31R (Figure 2B, rows 3–4 versus row 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)

Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
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Figure 2: Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.
Mentions: M18V strongly suppresses the dominant His+/Sui− phenotype, but not the recessive lethality, conferred by G31R. Thus, transformants harboring tif5-G31R,M18V fail to grow on −His medium in the presence of TIF5+, indicating the Ssu− phenotype, and also cannot grow on 5-FOA medium, indicating inviability of the cells following eviction of TIF5+ (Figure 2A, rows 3–4). The failure to grow on the −His medium does not result from a general growth defect, as the tif5-G31R,M18V strain grows better on +His medium than does the parental His+tif5-G31R strain, which displays the dominant Slg− phenotype conferred by G31R (Figure 2B, rows 3–4 versus row 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