Space-filling (left) and ribbon (right) representations of yeast eIF1 (pdb id 2ogh;25) showing the pocket created by G107 (red) surrounded by basic (blue) and hydrophobic (yellow) residues. Only part of the unstructured N-terminus (bottom right in structure) is shown.

(A) Binding of fluorescein-labeled eIF1 mutants was monitored using fluorescence anisotropy: (●) WT eIF1; (○) WT eIF1 + eIF1A; (■) G107K; (□) G107K + eIF1A; (▼) G107E; (∇) G107E + eIF1A. Each curve is the average of at least three experiments. (B) K_d values for eIF1 mutants binding to the 40S subunit. The ratio of K_ds in the absence and presence of eIF1A is the fold thermodynamic coupling between the two factors. Errors given are mean deviations.

(A–B) Sucrose gradient analysis of formaldehyde cross-linked WCEs from SUI1 (JCY145) and SUI1-G107K (JCY645) strains. Gradient fractions and 1% of each WCE (In), were subjected to Western analysis with antibodies against the indicated eIFs or 40S subunit protein RPS2. Fractions containing free 40S subunits are boxed. (B) Initiation factor binding to 40S subunits in the experiment in (A) and two replicates were quantified by calculating the ratios of eIF to RPS2 intensities in the 40S and normalizing the resulting mutant ratio by the corresponding WT ratio. The means ± SEs are plotted. The direction of sedimentation is from left to right. The lack of an increase in abundance of unbound factors in the gradients with the G107K mutant, which might have been expected given the loss of these proteins from the PICs, is attributed to increased instability of free eIFs relative to those in MFC or PICS in vivo or during sedimentation14. (C–D) eIF5 over-expression enhances the Sui⁻ phenotypes of eIF1 mutants. GCN2 his4-303 or GCN2 his4-303-myc strains described in Figs. 2A and C, respectively, were transformed with hc TIF5 plasmid (p4438) and analyzed for growth phenotypes (C) and His4-303-myc expression (D) as described in Fig. 2. (E–F). The G107K and -R mutations affect a functional interaction between eIF1 and eIF5. (E) The effect of eIF5 on TC binding to 40S subunits in the absence of mRNA with saturating eIF1 variants: WT, green; G107K, red; G107R, purple; G107E, blue. (F) The effect of saturating (2 μM) eIF5 on the kinetics of TC recruitment to the 40S subunit with eIF1 variants. The colors are the same as in E with open and closed symbols indicating the presence and absence of eIF5, respectively.

Renilla luciferase mRNA with either an AUG or UUG start codon was translated in WT yeast extracts supplemented with 2.8 μM WT or mutant eIF1. The mRNA with the UUG start codon is translated 10-fold less efficiently than the mRNA with the AUG codon in extracts with only WT eIF1. Luciferase activity was measured after 15 minutes (linear range). The reported values are normalized to the value for that mRNA with eIF1 storage buffer added to the extract instead of the additional eIF1; no change caused by the added WT or mutant eIF1 will give a normalized value of 1.0.

Figure 2. (A–C) Substitutions at G107 in eIF1 reduce stringent AUG selection in vivo. (A) Sui⁻ phenotypes of G107 mutations. GCN2 his4-303 strains with the indicated WT or mutant His-SUI1 alleles on sc or hc plasmids were grown at 30 °C for 2d on SC medium lacking leucine (+His; 120 μM histidine) and for 7d on SD medium plus uracil, tryptophan and 1.2 μM histidine (0.01X His). (B) Quantification of Sui⁻ phenotypes. Strains from (A) bearing plasmids p367 or p391 containing HIS4-lacZ fusions with an AUG or UUG start codon, respectively, were grown in SD with 0.3 mM His and Trp at 30 °C and β-galactosidase activities were measured in WCEs. Mean ratios of expression of UUG to AUG reporters are shown with standard errors (SE) from 3 experiments on 6 independent transformants. (C) Sui⁻ mutants increase myc-his4-303 expression. his4-303-myc strains harboring the indicated WT or mutant SUI1 alleles were grown overnight in YPD and WCEs were subjected to Western analysis with anti-myc, anti-eIF1, and anti-GCD6 antibodies. We do not yet know the nature of the slower migrating eIF1 band. (D–F) G107 substitutions partially derepress GCN4 translation. (D) gcn2Δ strains harboring sc His-SUI1 alleles were grown on SC-L medium at 30 °C for 2d, and on SC-L lacking histidine and containing 5 mM 3-AT for 4d. (E) The gcn2Δ strains described in (D) harboring the GCN4-lacZ reporter (with all 4 uORFs) were grown in SC-L, and β-galactosidase activities (nmol o-nitrophenyl-β-D-galactopyranoside cleaved min⁻¹ mg⁻¹) were measured in WCEs. The means and SEs from 3 or more measurements on 6 independent transformants are shown. (F) The GCN2 his4-303 strains with sc His-SUI1 alleles from (A) harboring GCN4-lacZ plasmid p180 were grown in SC lacking uracil, leucine, isoleucine and valine for 6h with or without sulfometuron 5 mg/ml) and β-galactosidase activities were measured in WCEs.

(A) The native gel-shift assay was used to measure TC affinity for the 40S subunit in the presence of saturating WT or mutant eIF1 and eIF1A in the absence of mRNA: (●) WT; (○) G107K; (■) G107R; (▲) G107S; (□) G107E; (△) R85S. (B – C) The kinetics of TC recruitment in the presence (B) and absence (C) of mRNA. The symbols are the same as in (A). The concentration of 40S subunits used was 10 nM in presence of mRNA and 200 nM in absence of mRNA. The curves shown are averages of three independent experiments. (D) Thermodynamic and kinetic constants for TC recruitment to the 40S•eIF1•eIF1A complex. TC binding in the absence of mRNA is biphasic indicating the existence of an additional step on the pathway10.

(A) eIF1 release was monitored by following the decrease in FRET (increase in fluorescein fluorescence) between eIF1-Fl and eIF1A-TAMRA in the PIC after addition of model mRNA with an AUG or AUU start codon. Curves were fit with double-exponential rate equations. The fast phase corresponds to a conformational change and the slow phase to eIF1 release. WT eIF1: AUG (green), AUU (blue); G107K: AUG (red), AUU (yellow); G107E: AUG (brown), AUU (black). (B) Comparison of rate constants for eIF1 release from 43S PICs with model mRNA with an AUG (green) or AUU (blue) start codon. (C) Rate constants for P_i release from PICs made with WT and mutant eIF1 in the absence and presence of model mRNAs with different start codons: AUG (green); AUU (blue); CUC (red); no mRNA (yellow). (D) The effect of eIF5 on the rate of release of WT and G107K eIF1 from 43S PICs with model mRNA with an AUG (green) or AUU (blue) start codon.

Prior to start codon recognition, the PIC is in an open conformation with relatively unstable TC binding. eIF1 interacts with the CTD of eIF5 and the NTD of eIF5 interacts with eIF2, activating it for GTP hydrolysis. When the start codon is encountered, codon-anticodon base pairing induces release of eIF1, which causes the PIC to undergo a conformational change into the closed state. eIF1 release stabilizes TC binding. In (A), release of eIF1 allows the NTD of eIF5 to enter the eIF1 binding site. In (B), eIF1 release allows the CTD of eIF5 to interact with eIF2β, which in turn causes movement of eIF5’s NTD. In either case, movement of eIF5’s NTD and/or a conformational change in eIF2 allows P_i release from eIF2 and establishment of the strong interaction between eIF5 and eIF1A. For simplicity, eIF2 is shown as a single protein instead of a trimer and the acceptor end of tRNA_i is shown free instead of bound to eIF2.