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- PDB-8bhl: Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in... -
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Basic information
Entry | Database: PDB / ID: 8bhl | ||||||
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Title | Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in the P-site and Am6AA mRNA codon in the A-site after uncompleted di-peptide formation | ||||||
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![]() | TRANSLATION / m6A / mRNA modification / tRNA recognition | ||||||
Function / homology | ![]() ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation ...ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / ribosomal large subunit assembly / transcription antitermination / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / molecular adaptor activity / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.21 Å | ||||||
![]() | Koziej, L. / Glatt, S. | ||||||
Funding support | European Union, 1items
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![]() | ![]() Title: Modulation of translational decoding by mA modification of mRNA. Authors: Sakshi Jain / Lukasz Koziej / Panagiotis Poulis / Igor Kaczmarczyk / Monika Gaik / Michal Rawski / Namit Ranjan / Sebastian Glatt / Marina V Rodnina / ![]() ![]() Abstract: N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how ...N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how mA modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of mA fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. mA does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 4.8 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 16059MC ![]() 8bf7C ![]() 8bgeC ![]() 8bghC ![]() 8bh4C ![]() 8bhjC ![]() 8bhnC ![]() 8bhpC ![]() 8bilC ![]() 8bimC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
+50S ribosomal protein ... , 28 types, 28 molecules ABCDEFGHIJKLMNOPQRSTUVWabcde
-30S ribosomal protein ... , 20 types, 20 molecules fghijklmnopqrstuvwxy
#29: Protein | Mass: 26781.670 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#30: Protein | Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#31: Protein | Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#32: Protein | Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#33: Protein | Mass: 15211.058 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#34: Protein | Mass: 20055.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#35: Protein | Mass: 14146.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#36: Protein | Mass: 14886.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#37: Protein | Mass: 11755.597 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#38: Protein | Mass: 13870.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#39: Protein | Mass: 13768.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#40: Protein | Mass: 13128.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#41: Protein | Mass: 11606.560 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#42: Protein | Mass: 10290.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#43: Protein | Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#44: Protein | Mass: 9724.491 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#45: Protein | Mass: 9005.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#46: Protein | Mass: 10455.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#47: Protein | Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#48: Protein | Mass: 8524.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-RNA chain , 5 types, 5 molecules 01234
#49: RNA chain | Mass: 941612.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#50: RNA chain | Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#51: RNA chain | Mass: 499690.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#52: RNA chain | Mass: 9739.893 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#53: RNA chain | Mass: 24802.785 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-Non-polymers , 1 types, 1 molecules ![](data/chem/img/FME.gif)
#54: Chemical | ChemComp-FME / |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight |
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Source (natural) |
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Buffer solution | pH: 7.5 / Details: 1x TAKM7 buffer | ||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Initiation complex containing: 1x TAKM7 buffer 1 mM DTT (dithiothreitol) 1 mM GTP (guanosine 5'-triphosphate) 2.0 uM 70S ribosomes 3.0 uM IF1, IF2, IF3 mix 6.0 uM A-site Am6AA mRNA 3.0 uM ...Details: Initiation complex containing: 1x TAKM7 buffer 1 mM DTT (dithiothreitol) 1 mM GTP (guanosine 5'-triphosphate) 2.0 uM 70S ribosomes 3.0 uM IF1, IF2, IF3 mix 6.0 uM A-site Am6AA mRNA 3.0 uM fMet-tRNAfMet Mixed 1:1 with a ternary complex containing: 1x TAKM7 buffer 3 mM PEP (phosphoenolpyruvate) 1 mM GTP (guanosine 5'-triphosphate) 1 mM DTT (dithiothreitol) 1% PK (pyruvate kinase) 0.02 uM EF-Ts 1.5 uM EF-Tu 0.3 uM Lys-tRNALys Incubated on ice for 60 seconds Final concentration of the sample: 20 A260 (absorbance at 260 nm, 10 mm path), 10 A280 (absorbance at 280 nm, 10 mm path) | ||||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.82 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7172 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 500 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 244504 / Algorithm: FOURIER SPACE / Num. of class averages: 9 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Refine LS restraints |
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