Japan Agency for Medical Research and Development (AMED)
21ae0121028h0001
Japan
Japan Society for the Promotion of Science (JSPS)
20H00451
Japan
Citation
Journal: Nat Commun / Year: 2023 Title: Dynamically regulated two-site interaction of viral RNA to capture host translation initiation factor. Authors: Shunsuke Imai / Hiroshi Suzuki / Yoshinori Fujiyoshi / Ichio Shimada / Abstract: Many RNA viruses employ internal ribosome entry sites (IRESs) in their genomic RNA to commandeer the host's translational machinery for replication. The IRES from encephalomyocarditis virus (EMCV) ...Many RNA viruses employ internal ribosome entry sites (IRESs) in their genomic RNA to commandeer the host's translational machinery for replication. The IRES from encephalomyocarditis virus (EMCV) interacts with eukaryotic translation initiation factor 4 G (eIF4G), recruiting the ribosomal subunit for translation. Here, we analyze the three-dimensional structure of the complex composed of EMCV IRES, the HEAT1 domain fragment of eIF4G, and eIF4A, by cryo-electron microscopy. Two distinct eIF4G-interacting domains on the IRES are identified, and complex formation changes the angle therebetween. Further, we explore the dynamics of these domains by using solution NMR spectroscopy, revealing conformational equilibria in the microsecond to millisecond timescale. In the lowly-populated conformations, the base-pairing register of one domain is shifted with the structural transition of the three-way junction, as in the complex structure. Our study provides insights into the viral RNA's sophisticated strategy for optimal docking to hijack the host protein.
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
JEOL CRYO ARM 300
Specialist optics
Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Details
The microscope model is the JEOL's "JEM-Z320FHC", the custom-built model equipped with a helium-cooled specimen stage.
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 6194 / Average exposure time: 8.0 sec. / Average electron dose: 71.1 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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