Translation initiation factor 2gamma mutant alters start codon selection independent of Met-tRNA binding

Abstract

Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNA(i)(Met) and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNA(i)(Met) to the 40S ribosomal subunit, and previous studies identified Sui(-) mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNA(i)(Met) binding. Consistently, an eIF2gamma-N135D GTP-binding domain mutation impairs Met-tRNA(i)(Met) binding and causes a Sui(-) phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNA(i)(Met) binding affinity and cell growth; however, only A208V suppresses the Sui(-) phenotype associated with the eIF2gamma-N135D mutation. An eIF2gamma-A219T mutation impairs Met-tRNA(i)(Met) binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui(-) phenotype associated with the eIF2gamma-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui(-) phenotype of the eIF2gamma mutants. We propose that structural alterations in eIF2gamma subtly alter the conformation of Met-tRNA(i)(Met) on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA(i)(Met) binding affinity.

FIG. 1.

Growth and Gcd⁻ and Sui⁻ phenotypes associated with the N135D mutation in Switch I of eIF2γ and its intragenic suppressors. (A) Schematic of eIF2γ. eIF2γ is divided into three domains based on sequence homology with structurally characterized EF-Tu and aIF2γ. The locations of conserved G domain sequence motifs G1/P-loop, G2, G3/DXXG, and G4/NKXD are indicated along with the locations of the structurally important and nucleotide-sensitive Switch I and Switch II elements. Above the schematic are sequence alignments around the sites of the eIF2γ-N135D mutation in Switch I and the suppressor mutations T115A, A208V, A219T, and A382V. The mutated residues in yeast (S. cerevisiae) eIF2γ are depicted in bold and aligned with the corresponding sequences from archaeal (Methanococcus jannaschii and Pyrococcus abyssi) aIF2γ, human eIF2γ, and Escherichia coli translation elongation GTPase EF-Tu. (B) Growth rate analysis of yeast expressing WT and mutant forms of eIF2γ. Derivatives of yeast strain J292 expressing the indicated WT or mutant form of eIF2γ from low-copy-number plasmids and carrying either a high-copy-number (H.C.) IMT4 plasmid expressing tRNA_i^Met (+; H.C. tRNA_i^Met; pC1683 [24]), or an empty vector (-) were grown to saturation, and 4-μl volumes of serial dilutions (with optical densities at 600 nm of 1.0, 0.1, 0.01, 0.001, and 0.0001) were spotted on minimal medium with essential nutrients (SD) and incubated for 3 days at 30°C. The doubling times during exponential growth in liquid SD medium are shown in parentheses. (C) Gcd⁻ phenotypes. A WT GCN4-lacZ reporter construct (p180) was introduced into the WT and mutant eIF2γ strains described for panel A and carrying either an empty high-copy-number vector or a high-copy-number plasmid expressing tRNA_i^Met . Transformants were grown to an A₆₀₀ of 0.7 in SD medium, whole-cell extracts were prepared, and β-galactosidase activity (nmol of o-nitrophenyl-β-d-galactopyranoside cleaved per min per mg) was measured for three independent transformants. Means and standard errors are plotted. For the H.C. vector data (black bars), P was <0.0002 for WT versus N135D and for N135D versus N135D,A208V or N135D,A219T; P was <0.003 for N135D versus A219T or N135D,A382V. (D) Sui⁻ phenotypes. HIS4^AUG-lacZ or his4^UUG-LacZ reporters were introduced into the strains described for panel C. β-Galactosidase activities were measured from one to four independent transformants and were used to calculate the mean UUG/AUG ratio ± the standard error. White and black bars denote transformants carrying a high-copy-number IMT4 plasmid and empty vector, respectively; gray bars denote transformants carrying neither the high-copy-number plasmid nor vector. For H.C. vector data (black bars), P was <0.003 for WT versus N135D, for N135D versus A219T, and for N135D versus N135D,A208V or N135D,A219T or N135D,A219T,A382V; P was ∼0.09 for N135D versus N135D,A382V.

FIG. 2.

Analysis of eIF2γ expression in vivo and purification of WT and mutant eIF2 complexes. (A) Western blot analysis of eIF2γ expression. Whole-cell extracts (20 and 40 μg) of strains described in Fig. 1B were subjected to immunoblot analysis using anti-yeast eIF2γ (upper panel, gray arrowhead) or anti-yeast eIF2Bɛ (GCD6) antiserum (lower panel, open arrowhead). Immune complexes were visualized using enhanced chemiluminescence, and the amount of eIF2γ was quantified relative to the eIF2Bɛ loading control. The amount of the eIF2γ mutants relative to WT (100%) is summarized above the lanes. (B) Purification of eIF2 complexes containing WT or mutant eIF2γ subunits. eIF2 complexes were purified from strains overexpressing eIF2α, eIF2β, eIF2γ, and His₈-eIF2γ (WT or mutant) as described in Materials and Methods. Two different amounts (0.5 and 2 μg) of the eIF2 complexes were resolved by 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with GelCode blue. The positions of the eIF2γ subunit (gray arrowhead) and eIF2α and eIF2β subunits (open arrowhead, doublet) are indicated.

FIG. 3.

Overexpression of eIF1 suppresses the Sui⁻ phenotype of eIF2γ mutants. (A) Growth rate analysis of yeast expressing WT or mutant forms of eIF2γ and overexpressing eIF1. Derivatives of yeast strain J292 expressing WT eIF2γ, eIF2γ-N135D, or eIF2γ-N135D, A382V and carrying either an empty vector (-), a high-copy-number (H.C.) plasmid expressing eIF1 (+, pC2888), or a high-copy-number plasmid expressing tRNA_i^Met (bottom two rows) were serially diluted, spotted on SD medium, and incubated for 3 days at 30°C as described for Fig. 1B. The doubling times during exponential growth in liquid SD medium are shown in parentheses. (B) Suppression of the Sui⁻ phenotype by overexpression of eIF1. Derivatives of yeast strain J292 expressing the indicated forms of eIF2γ and carrying either a high-copy-number plasmid expressing eIF1 or an empty vector were transformed with the HIS4^AUG-lacZ or his4^UUG-LacZ reporters, and β-galactosidase activities and UUG/AUG ratios were determined as described for Fig. 1D. For comparisons in the absence and presence of eIF1, P was ∼0.002 for WT, ∼0.006 for N135D,A208V, and <0.006 for N135D and for N135D,A382V. (C) The GCN4-lacZ reporter p180 was introduced into the strains described for panel B, and β-galactosidase activities were measured and plotted as described for Fig. 1C.

FIG. 4.

Models depicting the locations of eIF2γ mutations and their impacts on Met-tRNA_i^Met binding and start codon selection. (A) Model of eIF2γ mutations on the structure of the EF-Tu-GDPNP-Phe-tRNA complex. (1) Ribbon representation of the EF-Tu ternary complex (PDB ID code 1TTT), showing Phe-tRNA (orange) and GDPNP (cyan) bound to the EF-Tu. The EF-Tu G domain is depicted in brown, while domains 2 and 3 are marine blue and green, respectively. The lime sphere represents Mg²⁺. The side chains of key residues are depicted as sticks, with Asn135 in blue and the sites of suppressor mutations in green. The dashed box indicates the region shown in panels 2 and 3. (2) Model focusing on the locations of the N135D mutation (blue) in the Switch I element (warm pink), the A382V mutation (green) in domain 2, and the A219T mutation (green) in the G domain. (3) A different orientation of the structure in panel 2, rotated to reveal the location of the A208V suppressor mutation (green) in the Switch II element (magenta) of the G domain. (B) Hypothetical models depicting the impacts of eIF2γ mutations on Met-tRNA_i^Met binding and start codon selection. (1) In WT complexes, Met-tRNA_i^Met binds to eIF2 with high affinity (upward arrow in key below image) and in an optimum conformation that favors AUG codon recognition (base-pairing between the anticodon of the tRNA and the AUG codon of the mRNA. (2) The eIF2γ-N135D Switch I mutation impairs Met-tRNA_i^Met binding (pink; downward arrows in key) and lowers the stringency for AUG recognition (loss of base pair contact between the Met-tRNA_i^Met and the first nucleotide of the codon). The Met-tRNA_i^Met is bound in an altered conformation relative to its position on WT eIF2 (light gray). We propose that in this 48S complex eIF1 fails to properly monitor the codon-anticodon interaction and Met-tRNA_i^Met selects UUG as the start codon. (3) The A208V mutation in eIF2γ-N135D,A208V stabilizes Met-tRNA_i^Met binding (red; upward arrows in key) and restores the proper positioning of the Met-tRNA_i^Met in the 48S complex, resulting in a high-fidelity codon-anticodon interaction and suppression of the Sui⁻ phenotype. (4) The eIF2γ-A219T mutation weakens the affinity for Met-tRNA_i^Met (pink); however, the Met-tRNA_i^Met is positioned in the WT conformation, resulting in good codon-anticodon interactions and no Sui⁻ phenotype. (5) The A382V mutation in domain 2 of eIF2γ-N135D,A382V stabilizes Met-tRNA_i^Met binding (red); however, the Met-tRNA_i^Met is bound in an altered conformation, as observed with the original eIF2γ-N135D mutant. The improper position of Met-tRNA_i^Met in the 48S complex results in a failure of eIF1 to properly monitor the codon-anticodon interaction, and Met-tRNA_i^Met selects UUG as the start codon, resulting in a Sui⁻ phenotype.