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Entry
Database: PDB / ID: 5u4j
TitleStructural Basis of Co-translational Quality Control by ArfA and RF2 Bound to Ribosome
Components
  • (30S ribosomal protein ...) x 4
  • 16S rRNA
  • 23S rRNA23S ribosomal RNA
  • Alternative ribosome-rescue factor A
  • P-site tRNA fMet
  • Peptide chain release factor 2
  • mRNAMessenger RNA
KeywordsRIBOSOME / ArfA / RF2 / nonstop translation
Function / homology
Function and homology information


translation release factor activity, codon specific / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination / negative regulation of translational initiation / rescue of stalled ribosome / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing ...translation release factor activity, codon specific / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination / negative regulation of translational initiation / rescue of stalled ribosome / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / transcription antitermination / DNA-templated transcription termination / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / cytosolic small ribosomal subunit / regulation of translation / small ribosomal subunit / cytoplasmic translation / tRNA binding / ribosome / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / cytosol / cytoplasm
Similarity search - Function
Alternative ribosome-rescue factor A / Alternative ribosome-rescue factor A / Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Double Stranded RNA Binding Domain - #20 / Peptide chain release factor class I ...Alternative ribosome-rescue factor A / Alternative ribosome-rescue factor A / Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Double Stranded RNA Binding Domain - #20 / Peptide chain release factor class I / RF-1 domain / Ribosomal Protein S5; domain 2 - #10 / Ribosomal Protein S5; domain 2 / Double Stranded RNA Binding Domain / Nucleic acid-binding proteins / Ribosomal protein S3, bacterial-type / Ribosomal protein S4, bacterial-type / Ribosomal protein S5, bacterial-type / Ribosomal protein S12, bacterial-type / K Homology domain / K homology RNA-binding domain / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / KH domain / Type-2 KH domain profile. / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / K homology domain superfamily, prokaryotic type / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S5 / Ribosomal protein S5, N-terminal / Ribosomal protein S5, N-terminal domain / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, C-terminal / Ribosomal protein S5, C-terminal domain / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S4, conserved site / Ribosomal protein S4 signature. / Ribosomal protein S4/S9 / K homology domain-like, alpha/beta / S4 RNA-binding domain profile. / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / S4 RNA-binding domain / S4 domain / RNA-binding S4 domain / RNA-binding S4 domain superfamily / Ribosomal protein S12 signature. / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold / Beta Barrel / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
: / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Peptide chain release factor RF2 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS3 ...: / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Peptide chain release factor RF2 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Alternative ribosome-rescue factor A
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZeng, F. / Chen, Y. / Remis, J. / Shekhar, M. / Phillips, J.C. / Tajkhorshid, E. / Jin, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120552 United States
CitationJournal: Nature / Year: 2017
Title: Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.
Authors: Fuxing Zeng / Yanbo Chen / Jonathan Remis / Mrinal Shekhar / James C Phillips / Emad Tajkhorshid / Hong Jin /
Abstract: Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs ...Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.
History
DepositionDec 4, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2017Group: Database references
Revision 1.2Feb 8, 2017Group: Database references
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software / Item: _em_sample_support.grid_type / _em_software.name
Revision 1.5Oct 3, 2018Group: Data collection / Other / Refinement description / Category: cell / refine / Item: _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.6Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.7Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
a: 16S rRNA
A: 23S rRNA
c: 30S ribosomal protein S3
d: 30S ribosomal protein S4
e: 30S ribosomal protein S5
z: mRNA
x: P-site tRNA fMet
l: 30S ribosomal protein S12
v: Peptide chain release factor 2
w: Alternative ribosome-rescue factor A


Theoretical massNumber of molelcules
Total (without water)1,600,33210
Polymers1,600,33210
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 4 types, 4 molecules aAzx

#1: RNA chain 16S rRNA /


Mass: 497075.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 817573384
#2: RNA chain 23S rRNA / 23S ribosomal RNA


Mass: 941790.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 42756
#6: RNA chain mRNA / Messenger RNA


Mass: 5922.644 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: RNA chain P-site tRNA fMet


Mass: 24786.785 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: RNA was prepared by in vitro transcription / Source: (synth.) Escherichia coli (E. coli) / References: GenBank: 687670942

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30S ribosomal protein ... , 4 types, 4 molecules cdel

#3: Protein 30S ribosomal protein S3 /


Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Gene Name(s): rpsC b3314 JW3276 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7V3
#4: Protein 30S ribosomal protein S4 /


Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Gene Name(s): rpsD ramA b3296 JW3258 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7V8
#5: Protein 30S ribosomal protein S5 /


Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Gene Name(s): rpsE spc b3303 JW3265 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7W1
#8: Protein 30S ribosomal protein S12 /


Mass: 13768.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Gene Name(s): rpsL strA b3342 JW3304 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7S3

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Protein , 2 types, 2 molecules vw

#9: Protein Peptide chain release factor 2 / RF-2


Mass: 43299.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gene Name(s): prfB ECK2886 JW5847 supK / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MRE600 / Gene: prfB, supK, b2891, JW5847 / Production host: Escherichia coli (E. coli) / References: UniProt: P07012
#10: Protein Alternative ribosome-rescue factor A /


Mass: 6513.396 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gene Name(s): arfA ECK3278 JW3253 yhdL / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MRE600 / Gene: arfA, yhdL, b4550, JW3253 / Production host: Escherichia coli (E. coli) / References: UniProt: P36675

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Non-stop ribosomal complex with ArfA and RF2RIBOSOMEall0MULTIPLE SOURCES
230S subunitProkaryotic small ribosomal subunitCOMPLEX#1, #3-#5, #81NATURAL
350S subunitProkaryotic large ribosomal subunitCOMPLEX#21NATURAL
4Alternative ribosome-rescue factor AORGANELLE OR CELLULAR COMPONENT#101RECOMBINANT
5Peptide chain release factor 2ORGANELLE OR CELLULAR COMPONENT#91RECOMBINANT
6P-site tRNA fMetORGANELLE OR CELLULAR COMPONENT#71MULTIPLE SOURCESP-site tRNA (fMet) was in vitro transcribed.
7mRNAMessenger RNAORGANELLE OR CELLULAR COMPONENT#61MULTIPLE SOURCES
823S rRNA23S ribosomal RNAORGANELLE OR CELLULAR COMPONENT#23NATURAL
916S rRNAORGANELLE OR CELLULAR COMPONENT#12NATURAL
1030S ribosomal protein S3ORGANELLE OR CELLULAR COMPONENT#32NATURAL
1130S ribosomal protein S4ORGANELLE OR CELLULAR COMPONENT#42NATURAL
1230S ribosomal protein S5ORGANELLE OR CELLULAR COMPONENT2NATURAL
1330S ribosomal protein S12ORGANELLE OR CELLULAR COMPONENT2NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
21NO
31NO
416.35 kDa/nmNO
5140.13 kDa/nmNO
61NO
71NO
81NO
91
110
111
112
113
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
55Escherichia coli (E. coli)562
66Escherichia coli (E. coli)562
78Escherichia coli (E. coli)562
89Escherichia coli (E. coli)562
910Escherichia coli (E. coli)562
1011Escherichia coli (E. coli)562
1112Escherichia coli (E. coli)562
1213Escherichia coli (E. coli)562
11Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
34Escherichia coli (E. coli)562BL21pET28
45Escherichia coli (E. coli)562BL21pETNKI-his3C-LIC-Kan
56Escherichia coli (E. coli)562pUC18
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O4S1
215 mMmagnesium acetateMg(CH3COO)21
3150 mMpotassium acetateCH3CO2K1
44 mM2-mercapthoethanolHOCH2CH2SH1
52 mMspermidineC7H19N31
60.05 mMsperminC10H26N41
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/0.5 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Grids were blotted for 3.5 s

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 83822 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6 sec. / Electron dose: 20 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 30 / Used frames/image: 4-25

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Processing

SoftwareName: REFMAC / Version: 5.8.0155 / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
12RELION23D reconstruction
13REFMAC5.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155440 / Symmetry type: POINT
Atomic model buildingB value: 108.501 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: Average FSC
RefinementResolution: 3.7→458.11 Å / Cor.coef. Fo:Fc: 0.596 / SU B: 6.611 / SU ML: 0.094 / ESU R: 0.086
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.43355 --
obs0.43355 3976064 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 108.51 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å2-0.46 Å2-1.7 Å2
2--0.01 Å2-0.01 Å2
3---0.16 Å2
Refinement stepCycle: 1 / Total: 11360
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01412278
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg0.9041.51718210
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg3.8525517
ELECTRON MICROSCOPYr_dihedral_angle_2_deg41.52622.139187
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.79415776
ELECTRON MICROSCOPYr_dihedral_angle_4_deg8.7291550
ELECTRON MICROSCOPYr_chiral_restr0.0660.21954
ELECTRON MICROSCOPYr_gen_planes_refined0.0020.026637
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.19514.5572089
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it4.14921.8152599
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it1.2869.91610189
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined9.29320376
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork1.519 295394 -
Rfree-0 -
obs--100 %

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