Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability

Abstract

Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.

FIGURE 1.

Mutational analysis of helix H12 in yeast eIF5B. (A) Ribbon representation of X-ray crystal structure of aIF5B (M. thermoautotrophicum) in complex with GTP (Protein Data Bank [PDB] code 1G7T) (Roll-Mecak et al. 2000). The four domains of the protein (G, II, III, and IV) and helices (H6 and H12) are labeled. The structure on the right is rotated about the vertical axis to better visualize the lever-type domain IV movement in the presence of GTP (colored) versus GDP (gray; PDB code 1G7S). The inset in the blue box highlights the interactions between top of H12 and the G domain and domain III in aIF5B (labels refer to M. thermoautotrophicum residues). The inset in the red box depicts the corresponding H12 region in yeast (S. cerevisiae) eIF5B that was subjected to mutational analysis. (B) Sequence alignments of helix H12 region of eukaryotic, bacterial, and archaeal eIF5B/IF2. Identical residues within the aligned sequences are shown in dark green, conserved residues are shown in yellow green, and weakly conserved residues (conserved in three out of five residues sequences) are in yellow. Residue numbers are shown for S. cerevisiae and M. thermoautotrophicum, and the position of helix H12 is denoted by the black bar above the sequences. (C) Schematic representation of helix H12 mutants. Open boxes indicate deleted residues, whereas amino acid residues within the boxes indicate insertions (Plus3) or substitutions (Swap5 and 6Gly). Doubling times in SD medium of yeast strain J111 expressing the indicated H12 mutant are shown on the right. (D) Western blot analysis of eIF5B expression. Whole cell extracts prepared from yeast transformants in panel C were subjected to immunoblot analysis using anti-eIF5B or anti-eIF2α antiserum as previously described (Choi et al. 2000).

FIGURE 2.

Translational control of GCN4 expression in eIF5B mutant strains. (A) Growth phenotypes. Derivatives of the ΔeIF5B strain J111 transformed with plasmids expressing wild-type eIF5B (WT), empty vector (ΔeIF5B) or various H12 mutants, as indicated, were grown to saturation, and 5 μl of serial dilutions (of OD₆₀₀ = 1.0, 0.1, 0.01, 0.001, and 0.0001) were spotted on minimal medium supplemented with essential nutrients (SD) or medium containing 10 mM 3-aminotriazole (3-AT). Plates were incubated 3 d at 30°C. (B) Analysis of GCN4-lacZ expression. The wild-type GCN4-lacZ plasmid p180 (Hinnebusch 1985) or a derivative in which an extended version of uORF1 overlaps the GCN4 AUG start codon (pM226) (Grant et al. 1994) were introduced into derivatives of strain J111 described in panel A. Cells were grown and β-galactosidase activities were determined as described previously (Hinnebusch 1985). R, cells were grown under nonstarvation conditions in SD medium where GCN4 expression is repressed; DR, cells were grown under amino acid starvation conditions (SD + 10 mM 3-aminotriazole) where GCN4 expression is derepressed. The β-galactosidase activities are the averages of three independent transformants and have standard errors of 20% or less.

FIGURE 3.

Analysis of GTP and ribosome binding and GTPase activities of eIF5B mutant proteins. (A) K_d values and standard deviations for GTP binding to eIF5B mutants from three independent measurements. (B) Ribosome binding assay. Purified WT or helix H12 mutants of eIF5B were mixed in the presence of GTP or GDP, as indicated, with purified yeast 80S ribosomes, and then loaded onto a 10% sucrose cushion. Following centrifugation, the amounts of eIF5B recovered in the pellet and supernatant fractions were determined by SDS-PAGE and quantitative densitometry. The data presented represent the fraction of eIF5B present in the ribosomal pellet and are representative of results from at least three independent experiments ± standard error. (C) Ribosome-dependent GTPase assay. As shown in the reaction scheme (top), 1 μM eIF5B was incubated with 0.4 μM 80S ribosomes for 2 min. Following addition of 50 nM [γ-³³P]GTP, aliquots from the reactions were analyzed at the indicated times by thin-layer chromatography. The data presented are representative of results from at least three independent experiments. Data were fit to the single exponential expression A[1 − exp(−kt)], in which A is the amplitude, k is the rate constant, and t is time. Fits were performed using KaleidaGraph (Synergy Software), and values in parentheses are fit errors.

FIGURE 4.

Helix H12 mutants of eIF5B impair 80S complex stability. (A) Experimental scheme for subunit joining monitored by light scattering. (B) Subunit joining assay for WT eIF5B or the Swap5, 6Gly, or D6 mutant in the presence of GTP. Observed rate constant (k_obs) and amplitude for each reaction are shown at the right end of the corresponding curve. Data are means ± standard error from at least two independent experiments. (C) Experimental scheme for 80S complex formation assay. (D,E) Results from 80S complex formation assay. 48S complexes were formed using limiting amounts of eIF2 ternary complexes containing [³⁵S]Met-tRNA_i^Met and then subunit joining was monitored in the presence of either GTP (left panel) or GDPNP (right panel). Reactions were stopped at 15 and 30 min and loaded onto a running native gel. Following phosphorimage analysis of the gel (D), the amounts of [³⁵S]Met-tRNA_i^Met that were free or bound to 43S or 80S complexes at 30 min were quantified, and the fraction of [³⁵S]Met-tRNA_i^Met in 80S complexes was calculated (E). Data represent the mean of three independent experiments ± standard error.