- EMDB-48479: Damaged 70S ribosome with PrfH bound -
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Basic information
Entry
Database: EMDB / ID: EMD-48479
Title
Damaged 70S ribosome with PrfH bound
Map data
cryo-EM map of damage 70S E. coli Ribosome rescured by C. gingivalis PrfH
Sample
Complex: Damaged 70S with PrfH
Complex: 50S
RNA: x 2 types
Protein or peptide: x 31 types
Complex: 30S
RNA: x 1 types
Protein or peptide: x 20 types
Organelle or cellular component: tRNA
RNA: x 1 types
Organelle or cellular component: Putative peptide chain release factor homolog
Protein or peptide: x 1 types
RNA: x 1 types
Ligand: x 3 types
Keywords
CgPrfH / damaged ribosome / ribotoxin / RIBOSOME
Function / homology
Function and homology information
negative regulation of cytoplasmic translational initiation / stringent response / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation ...negative regulation of cytoplasmic translational initiation / stringent response / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / transcription antitermination / translational initiation / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / large ribosomal subunit / transferase activity / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm Similarity search - Function
Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A ...Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / : / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L9 signature. / : / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / : / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein S19, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily Similarity search - Domain/homology
Small ribosomal subunit protein bS18 / Large ribosomal subunit protein bL36 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL28 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS13 / Large ribosomal subunit protein bL20 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS3 / Large ribosomal subunit protein bL34 ...Small ribosomal subunit protein bS18 / Large ribosomal subunit protein bL36 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL28 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS13 / Large ribosomal subunit protein bL20 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS3 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL33 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS10 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein uL29 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL24 / Small ribosomal subunit protein uS8 / Large ribosomal subunit protein uL30 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS16 / Large ribosomal subunit protein bL32 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein bL21 Similarity search - Component
Biological species
Escherichia coli (E. coli) / Capnocytophaga gingivalis (bacteria)
Method
single particle reconstruction / cryo EM / Resolution: 2.65 Å
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM145060
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM120764
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM120552
United States
National Natural Science Foundation of China (NSFC)
32171200
China
Citation
Journal: bioRxiv / Year: 2025 Title: Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation. Authors: Yannan Tian / Qingrong Li / Shirin Fatma / Junyi Jiang / Hong Jin / Fuxing Zeng / Raven H Huang / Abstract: In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as ...In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as well as several alternative rescue factors (Arfs) are responsible for rescuing the stalled ribosomes caused by truncated mRNAs that lack the stop codons. To date, protein release factor homolog (PrfH) is the only factor known to rescue the stalled ribosome damaged by ribotoxins. Here we show that a subfamily of PrfH, exemplified by PrfH from (PrfH), rescues both types of stalled ribosomes described above. Our biochemical assays demonstrate that PrfH hydrolyzes the peptides attached to P-site tRNAs when in complex with both the damaged and intact ribosomes. Two cryo-EM structures of PrfH in complex with the damaged and intact 70S ribosomes revealed that PrfH employs two different regions of the protein to recognize two different stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization described here, we have uncovered a family of ribosome rescue factors that possesses dual activities to resolve two distinct stalled protein translation in bacteria.
Model: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.4 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 30.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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