National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM136936
United States
Robert A. Welch Foundation
H-2032-20200401
United States
Citation
Journal: Nat Commun / Year: 2022 Title: Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation. Authors: Ritwika S Basu / Michael B Sherman / Matthieu G Gagnon / Abstract: During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNA) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 ...During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNA) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (P) release, changes conformation facilitating fMet-tRNA accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNA accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.
EMPIAR-11012 (Title: Pseudomonas aeruginosa 70S ribosome initiation complex bound to compact IF2 Data size: 1.7 TB Data #1: Unaligned multiframe micrographs of the Pseudomonas aeruginosa 70S ribosome initiation complex bound to IF2 collected on Gatan K3 in Super Resolution [micrographs - multiframe])
Entire : Focused map of the N2 sub-domain of IF2 bound to the 30S subunit ...
Entire
Name: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
Components
Complex: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex
Protein or peptide: Translation initiation factor IF-2
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Supramolecule #1: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit ...
Supramolecule
Name: Focused map of the N2 sub-domain of IF2 bound to the 30S subunit in the 70S ribosome initiation complex type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Cryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 295 K / Instrument: LEICA EM GP
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Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8056 / Average exposure time: 1.0 sec. / Average electron dose: 31.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 123146
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classification
Software - Name: cryoSPARC (ver. 3.1.0)
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Atomic model buiding 1
Refinement
Space: REAL / Protocol: RIGID BODY FIT
Output model
PDB-7uiu: N2 sub-domain of IF2 bound to the 30S subunit in the Pseudomonas aeruginosa 70S ribosome initiation complex (focused classification and refinement)
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