Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition
- MRC Laboratory of Molecular Biology, United Kingdom
- Instituto de Biomedicina de Valencia (IBV-CSIC), Spain
- Indian Institute of Science, India
- Shoolini University of Biotechnology and Management Sciences, India
- Eunice K Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
Figures
Figure 1 with 6 supplements
Cryo-EM structure of py48S-eIF5N.
(A) CryoEM maps of the PIC py48S-eIF5N shown in three orientations. Regions of the map are colored by component to show the 40S subunit (yellow), eIF1A (blue), eIF5-NTD (cyan), Met-tRNAiMet (green), …
Figure 1—figure supplement 1
Scheme of 3D classification of data.
For Sample 1 (without crosslinking dataset) 394,672 particles were selected after 2D classification and an initial 3D refinement was done. After two rounds of 3D-classification, a class containing …
Figure 1—figure supplement 2
Validation of the maps.
(A) Gold-standard Fourier Shell Correlation (FSC) curves for the Maps 1, A and B. (B) Gold-standard Fourier Shell Correlation (FSC) curves for Maps C1 and C2. (C) Analysis of overfitting by …
Figure 1—figure supplement 3
Fitting of ligands in density maps.
(A) Fitting of mRNA (magenta), eIF1A (marine), eIF5-NTD (cyan), tRNAi (green), eIF2α (purple), eIF2β (red) and eIF2γ (orange) in Map C1. (B) As in A, but shown in a different orientation and …
Figure 1—figure supplement 4
Map quality and local resolution Surface (left or top) and cross-sections (right or bottom) of gaussian-filtered maps, colored according to local resolution.
(A) Map 1 (B) Map C1 (C) Map B (D) Map A.
Figure 1—figure supplement 5
Latch and h28 conformation and head closure in different py48S PICs.
Superimposition of different py48S complexes shows a different degree of head closure around the latch area (left inset). Right inset shows distinct h28 conformations; only py48S-open presents a …
Figure 1—figure supplement 6
Comparison of the maps obtained with particles from sample 1 (non-crosslinked) and sample 2 (1%-formaldehyde crosslinked).
(A,B) Particles in map B belonging to sample 1 (non-crosslinked; gray) or sample 2 (crosslinked; yellow) were refined independently. Resulting maps, shown separately in (A), or superposed (B) are …
Figure 2 with 3 supplements
Contacts of eIF5-NTD with the other components in the 48S PIC.
(A) A detailed view of the contacts of eIF5-NTD near the P site with the 40S subunit, tRNAi, mRNA, eIF1A and eIF2β. eIF5 residues involved in the contacts are shown in sticks. (B) Fitting of …
Figure 2—figure supplement 1
eIF5 and eIF1 comparison.
(A) Cartoon representation of eIF5 (this study). Secondary structure elements are labeled. (B) Cartoon representation of eIF1 (from PDB:3J81) with labeled secondary structure elements. (C) Amino …
Figure 2—figure supplement 2
Density of β-hairpins 1 and 2 of eIF5-NTD and its contacts with tRNAi.
(A) Fitting of β-hairpin 1 of eIF5-NTD (cyan) and tRNAi (green) in Map 1. (B) As in A, but for β-hairpin 2 of eIF5-NTD.
Figure 2—figure supplement 3
Comparison of eukaryotic and bacterial initiation following start-codon recognition.
(A) Accommodation of tRNAi in the P site in different yeast PICs. Similar representation to that in Figure 2C but including eIF5B and its associated tRNA (in gray) from the structure of an 80S …
Figure 3 with 2 supplements
Genetic evidence that contacts of the eIF5-NTD with the tRNAi ASL and mRNA AUG codon in py48S-eIF5N are crucial for stringency of start-codon recognition in vivo.
(A) Location of the eIF5 residues substituted in genetic studies, highlighted in red and shown as spheres. (B) Summary of eIF5-NTD residues substituted by TIF5 mutations (col. 1), their interactions …
Figure 3—figure supplement 1
Genetic assays for mutations altering the accuracy of start-codon selection in vivo.
(A–D) Schema depicting wild-type HIS4 mRNA (A) and his4-303 mRNA containing an ACG replacement of the AUG start codon, which diminishes translation of the HIS4-encoded histidine biosynthetic enzyme …
Figure 3—figure supplement 2
Genetic assays and eIF5 variants expression.
(A) Strains ASY101, PMY30 and PMY33 with the indicated relevant genotypes (rows 1–3, respectively) were spotted on SC-L medium supplemented with 0.3 mM histidine (+His) or 0.0003 mM histidine (1% …
Figure 4
eIF5-NTD Sui- mutants stabilize the closed conformation of the PIC at UUG codons.
Dissociation of fluorescein-tagged eIF1A from 43S·mRNA complexes reconstituted with model mRNAs containing an AUG or UUG start codon and either WT or Sui- variants of eIF5 was monitored as decrease …
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Figure 4—source data 1
Kinetic parameters for dissociation of eIF1A from 48S PIC.
- https://doi.org/10.7554/eLife.39273.021
Figure 5
eIF5-NTD Ssu- mutants destabilize the closed conformation of the PIC and accelerate Pi release at UUG codons in the presence of the SUI3-2 Sui- variant of eIF2.
(A–D) eIF1A dissociation kinetics experiments conducted as in Figure 4 for PICs assembled with mRNAs containing AUG (closed circles) or UUG (closed squares) start codons and the following forms of …
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Figure 5—source data 1
eIF1A dissociation kinetics assays.
- https://doi.org/10.7554/eLife.39273.024
Figure 6
P site conformation and surrounding elements in py48S-eIF5N.
(A) Cartoon representation of the TC in Maps C1 and C2, resulting from superposition of the 40S body in the two maps. The tRNAi, mRNA, and each component of TC is colored differently for C1, whereas …
Figure 7
mRNA path at the exit and entry of the 40S mRNA channel in py48S-eIF5N.
(A) Cross-section of the 40S subunit along the mRNA path of py48S-eIF5N, viewed from the top of the 40S subunit, showing both the entry and exit openings of the 40S mRNA channel. Path of the mRNA …
Figure 8
eIF3 architecture within py48S-eIF5N.
(A) Two different views of the py48S-eIF5N PIC showing the locations of the different eIF3 subunits. All eIF3 domains, except for the eIF3c N-terminal helical bundle, reside on the solvent-exposed …
Figure 9
eIF3 relocates back to the solvent-exposed surface of 40S.
(A) Modeling of the eIF3b/eIF3i/eIF3g/eIF3a-Cterm quaternary complex observed in py48S-closed-eIF3 (PDB 6GSN) at the subunit interface of py48S-eIF5N. The location of eIF5-NTD in the latter complex …
Figure 10
Schematic model of major conformational changes during initiation.
(I) Binding of eIF1, eIF1A and eIF3 to the 40S subunit facilitates TC binding in the POUT conformation to form the 43S PIC. The disordered NTT of eIF1A is shown as a dashed line. eIF2β and 3 c …
Videos
Video 1
Movie highlighting tRNA accommodation, leading to eIF1 dissociation and eIF5-NTD recruitment to the 48S complex.
Detailed contacts of eIF5-NTD with other elements of the 48S complex are also shown.
Tables
Table 1
Refinement and model statistics.
https://doi.org/10.7554/eLife.39273.013| Model with TC in conformation 1 | Model with TC in conformation 2 | |
|---|---|---|
| Model Composition | ||
| Non-hydrogen atoms | 104,332 | 104,232 |
| Protein residues | 8538 | 8522 |
| RNA bases | 1882 | 1882 |
| Refinement | ||
| Resolution used for refinement (Å) | 3.05 | 3.05 |
| Map sharpening B-factor (Å) | −67 | −66 |
| Average B-factor (Å) | 162 | 121 |
| Fourier Shell Correlation (FSC)* | 0.90 | 0.89 |
| Rms deviations | ||
| Bonds (Å) | 0.006 | 0.006 |
| Angles (°) | 1.139 | 1.194 |
| Validation (proteins) | ||
| Molprobity score (Percentile in brackets) | 2.65 (91st) | 2.74 (89th) |
| Clashscore, all atoms (Percentile in brackets) | 6.87 (100th) | 7.11 (98th) |
| Good rotamers (%) | 91.1 | 89.2 |
| Ramachandran plot | ||
| Favored (%) | 89.8 | 89.2 |
| Outliers (%) | 2.5 | 2.7 |
| Validation (RNA) | ||
| Correct sugar puckers(%) | 98.6 | 96.9 |
| Good backbone conformations(%) | 63.6 | 63.1 |
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*FSC= Σ(Nshell FSCshell)/ Σ(Nshell), where FSCshell is the FSC in a given shell, Nshell is the number of ‘structure factors’ in the shell. FSCshell = Σ(Fmodel FEM)/ (√(Σ(|F|2model)) √(Σ(|F|2EM)))
Table 2
Local resolution of ligands.
https://doi.org/10.7554/eLife.39273.014| Structures | Map 1 (Å) | Map A (Å) | Map B (Å) | Map C1 (Å) | Map C2 (Å) |
|---|---|---|---|---|---|
| Overall Resolution | 3.00 | 3.50 | 3.50 | 3.50 | 3.10 |
| eIF5-NTD | 3.15 | nd | nd | 3.80 | 3.35 |
| eIF1A | 3.00 | nd | nd | 3.70 | 3.10 |
| eIF2α | 3.65 | nd | nd | 4.15 | 3.65 |
| eIF2β | nd* | nd | nd | 9.40 | >15 |
| eIF2γ | 8.15 | nd | nd | 8.00 | 8.10 |
| tRNAi | 3.20 | nd | nd | 3.65 | 3.20 |
| ASL + mRNA(−4 to + 4) | 2.95 | nd | nd | 3.45 | 3.15 |
| eIF3 PCI domains | nd | 7.80 | nd | nd | nd |
| eIF3b/eIF3a-cterm | nd | nd | 7.25 | nd | nd |
| eIF3b-cterm/eIF3i/eIF3g | nd | nd | 12.20 | nd | nd |
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*nd – not determined
Table 3
Contribution of non-crosslinked (157,868 particles) and crosslinked (113,838 particles) datasets to each map.
https://doi.org/10.7554/eLife.39273.015| Structures Number of particles (% of the total) and resolution in Å | Map 1 | Map A | Map B | Map C1 | Map C2 |
|---|---|---|---|---|---|
| Non-crosslinked | 157,868 (58%) ; 3.00 | 23,219 (8.5%) ; 3.70 | 25,761 (9.5%) ; 3.60 | 27,012 (10%) ; 3.60 | 99,229 (36.5%) ; 3.10 |
| Crosslinked | - | 30,651 (11.5%) ; 4.30 | 28,938 (10.5%) ; 4.35 | 47,760 (17.5%) ; 4.30 | 37,874 (13.5%) ; 4.05 |
| Merged | 157,868 (58%) ; 3.00 | 53,870 (20%) ; 3.50 | 54,699 (20%) ; 3.50 | 74,772 (27.5%) ; 3.50 | 137,103 (50%) ; 3.10 |
Table 4
Kinetic parameters for dissociation of eIF1A from 48S PIC
https://doi.org/10.7554/eLife.39273.022| (A) Kinetic parameters for dissociation of eIF1A from 48S PIC in presence of eIF5 Sui- mutants | |||||||
|---|---|---|---|---|---|---|---|
| eIF5 variants | mRNA | k1 (open) (x10−3 s−1) | k2 (closed) (x10−3 s−1) | a1 (open) | a2 (closed) | Kamp* (a2/a1) | Rbound† |
| WT | AUG | 6 ± 1 | 0.4 ± 0.05 | 0.15 ± 0.02 | 0.85 ± 0.02 | 5.9 | 0.2105 ± 0.002 |
| UUG | 22 ± 4 | 2.1 ± 0.3 | 0.24 ± 0.02 | 0.76 ± 0.02 | 3.2 | 0.1820 ± 0.002 | |
| G31R | AUG | 18 ± 3 | 3.0 ± 0.4 | 0.33 ± 0.03 | 0.67 ± 0.03 | 2.1 | 0.1915 ± 0.001 |
| UUG | 7.0 ± 1.5 | 0.5 ± 0.02 | 0.13 ± 0.01 | 0.87 ± 0.02 | 6.9 | 0.2025 ± 0.001 | |
| N30R | AUG | 10.0 ± 1.0 | 1.5 ± 0.3 | 0.50 ± 0.1 | 0.50 ± 0.1 | 1.0 | 0.2030 ± 0.002 |
| UUG | 6.0 ± 1.0 | 0.5 ± 0.1 | 0.30 ± 0.05 | 0.70 ± 0.05 | 2.3 | 0.2100 ± 0.002 | |
| G29R | AUG | 20 ± 3.0 | 1.5 ± 0.4 | 0.40 ± 0.02 | 0.60 ± 0.02 | 1.5 | 0.1885 ± 0.001 |
| UUG | 6.0 ± 1.6 | 0.6 ± 0.1 | 0.20 ± 0.03 | 0.80 ± 0.03 | 4.0 | 0.1925 ± 0.002 | |
| E26K | AUG | 17 ± 2.6 | 0.9 ± 0.05 | 0.23 ± 0.02 | 0.77 ± 0.02 | 3.5 | 0.1835 ± 0.001 |
| UUG | 8.0 ± 1.5 | 0.4 ± 0.05 | 0.16 ± 0.01 | 0.84 ± 0.01 | 5.2 | 0.1900 ± 0.002 | |
| (B) Kinetic parameters for dissociation of eIF1A from 48S PIC in presence of eIF5 Ssu- and Sui3-2 eIF2 | |||||||
|---|---|---|---|---|---|---|---|
| eIF5 variants; Sui 3–2 eIF2 | mRNA | k1(open) (x10−3 s−1) | k2(closed) (x10−3 s−1) | a1(open) | a2(closed) | Kamp* (a2/a1) | Rbound† |
| Sui3-2 | AUG | 4 ± 1 | 0.3 ± 0.04 | 0.22 ± 0.03 | 0.78 ± 0.03 | 3.4 | 0.1945 ± 0.002 |
| UUG | 5 ± 1.8 | 0.5 ± 0.04 | 0.20 ± 0.02 | 0.80 ± 0.02 | 4.0 | 0.1910 ± 0.001 | |
| N30E | AUG | 6 ± 0.5 | 0.4 ± 0.04 | 0.15 ± 0.02 | 0.85 ± 0.01 | 5.6 | 0.2045 ± 0.0005 |
| UUG | 15 ± 4 | 1.0 ± 0.16 | 0.23 ± 0.01 | 0.77 ± 0.01 | 3.3 | 0.1830 ± 0.003 | |
| G29E | AUG | 8.0 ± 1.0 | 0.4 ± 0.02 | 0.22 ± 0.02 | 0.78 ± 0.02 | 3.5 | 0.2084 ± 0.0006 |
| UUG | 18 ± 3.0 | 0.36 ± 0.03 | 0.28 ± 0.02 | 0.72 ± 0.02 | 2.6 | 0.1830 ± 0.003 | |
| R28E | AUG | 13 ± 4.0 | 0.5 ± 0.04 | 0.21 ± 0.01 | 0.80 ± 0.01 | 4.0 | 0.2040 ± 0.001 |
| UUG | 23 ± 2.0 | 0.9 ± 0.06 | 0.25 ± 0.03 | 0.75 ± 0.03 | 3.0 | 0.1910 ± 0.003 | |
| R28A | AUG | 8.0 ± 0.8 | 0.3 ± 0.04 | 0.20 ± 0.02 | 0.80 ± 0.02 | 4.0 | 0.2105 ± 0.001 |
| UUG | 14 ± 4.0 | 0.7 ± 0.08 | 0.24 ± 0.01 | 0.76 ± 0.01 | 3.2 | 0.1810 ± 0.003 | |
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*Higher values of Kamp indicate that a greater proportion of the complexes are in the closed state (Saini et al., 2014).
†Higher values of Rbound indicate that more complexes are in the constrained, closed state ( Saini et al., 2014).
Table 5
Kinetic parameters for Pi release from 48S PIC.
https://doi.org/10.7554/eLife.39273.025| (A) Kinetic parameters for Pi release from 48S PIC with eIF5 Sui- mutants. | ||
|---|---|---|
| eIF5 Variants | Rate of Pi release (s−1) AUG UUG | |
| WT | 0.60 ± 0.08 | 0.26 ± 0.04 |
| G31R | 0.30 ± 0.04 | 0.60 ± 0.04 |
| N30R | 0.16 ± 0.02 | 0.41 ± 0.03 |
| G29R | 0.25 ± 0.03 | 0.55 ± 0.05 |
| E26K | 0.50 ± 0.20 | 0.72 ± 0.06 |
| (B) Kinetic parameters for Pi release from 48S PIC with eIF5 Ssu- mutants in presence of Sui3-2 eIF2 | ||
|---|---|---|
| eIF5 Variants; Sui3-2 eIF2 | Rate of Pi release (s−1) AUG UUG | |
| WT | 0.60 ± 0.10 | 0.75 ± 0.09 |
| N30E | 0.55 ± 0.15 | 0.30 ± 0.03 |
| G29E | 0.72 ± 0.04 | 0.40 ± 0.01 |
| R28A | 0.60 ± 0.02 | 0.35 ± 0.04 |
Additional files
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Supplementary file 1
Plasmids used in this study
- https://doi.org/10.7554/eLife.39273.031
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Supplementary file 2
Yeast strains used in this study
- https://doi.org/10.7554/eLife.39273.032
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Transparent reporting form
- https://doi.org/10.7554/eLife.39273.033
