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- EMDB-1391: Structured mRNAs regulate translation initiation by binding to th... -

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Basic information

Entry
Database: EMDB / ID: EMD-1391
TitleStructured mRNAs regulate translation initiation by binding to the platform of the ribosome.
Map dataCryo-EM map of the E.coli 70S pre-initiation complex elucidating the entrapment mechanism by the auto-regulatory S15-rpsOmRNA.
Sample
  • Sample: 70S pre-initiation complex entrapped by S15-rpsOmRNA
  • Complex: 70S
  • RNA: rpsO messanger RNA
  • Protein or peptide: Ribosomal protein S15
Function / homology
Function and homology information


mRNA base-pairing translational repressor activity / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / cytosolic small ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / translation / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein S15, bacterial-type / Ribosomal protein S15, bacterial-type / Ribosomal_S15 / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal protein S15 / S15/NS1, RNA-binding
Similarity search - Domain/homology
Small ribosomal subunit protein uS15
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 10.0 Å
AuthorsMarzi S
CitationJournal: Cell / Year: 2007
Title: Structured mRNAs regulate translation initiation by binding to the platform of the ribosome.
Authors: Stefano Marzi / Alexander G Myasnikov / Alexander Serganov / Chantal Ehresmann / Pascale Romby / Marat Yusupov / Bruno P Klaholz /
Abstract: Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ...Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.
History
DepositionJun 22, 2007-
Header (metadata) releaseJun 26, 2007-
Map releaseNov 15, 2007-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
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  • Surface view colored by height
  • Surface level: 1
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  • Surface view with fitted model
  • Atomic models: PDB-2vaz
  • Surface level: 1
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2vaz
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1391.gif

Downloads & links

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Map

FileDownload / File: emd_1391.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the E.coli 70S pre-initiation complex elucidating the entrapment mechanism by the auto-regulatory S15-rpsOmRNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 120 pix.
= 360. Å		3 Å/pix.
x 120 pix.
= 360. Å		3 Å/pix.
x 120 pix.
= 360. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.333
CCP4 map header333
EM Navigator Movie #12.42.42.4
Density

Histogram

Histogram (log scale)
Contour Level1: 2.33 / Movie #1: 1
Minimum - Maximum-9.050319999999999 - 20.798400000000001
Average (Standard dev.)0.140196 (±1.23037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-9.05020.7980.140

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Supplemental data

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Sample components

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Entire : 70S pre-initiation complex entrapped by S15-rpsOmRNA

EntireName: 70S pre-initiation complex entrapped by S15-rpsOmRNA
Components
  • Sample: 70S pre-initiation complex entrapped by S15-rpsOmRNA
  • Complex: 70S
  • RNA: rpsO messanger RNA
  • Protein or peptide: Ribosomal protein S15

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Supramolecule #1000: 70S pre-initiation complex entrapped by S15-rpsOmRNA

SupramoleculeName: 70S pre-initiation complex entrapped by S15-rpsOmRNA / type: sample / ID: 1000
Details: 70S E. coli ribosome complexed with S15-rpsOmRNA,stalling Translation Initiation at the pre-initiation step. The structure reveals the molecular details of the Entrapment mechanism.
Oligomeric state: monomer / Number unique components: 3
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: 70S

SupramoleculeName: 70S / type: complex / ID: 1 / Name.synonym: 70S / Details: E. coli 70S ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: rpsO messanger RNA

MacromoleculeName: rpsO messanger RNA / type: rna / ID: 1 / Name.synonym: S15mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
GGGCGAAUUC GAGCUCGGUA CCCAACGUCG CGUAAAUUGU UUAACACUUU GCGUAACGUA CACUGGGAUC GCUGAAUUAG AGAUCGGCGU CCUUUCAUUC UAUAUACUAA GGAGGUUAAA AUGUCUCUAA GUACUGAAGC AACAGCUAAA AUCGUUUCUG AGUUUGGUCG ACCUGCAGGC AUGCA

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Macromolecule #2: Ribosomal protein S15

MacromoleculeName: Ribosomal protein S15 / type: protein_or_peptide / ID: 2 / Name.synonym: S15 / Details: rpsO gene / Oligomeric state: monomeric / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET11c
SequenceGO: translation / InterPro: Ribosomal protein S15, bacterial-type

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 60 mM KCl, 1mM DTT, 7.5 mM MgCl2
StainingType: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids
GridDetails: 300 mesh Cu/Rh
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51484 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 77 K
Alignment procedureLegacy - Astigmatism: lens astigmatism was corrected at 50,000 times magnification
DateJan 5, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5 µm / Number real images: 67 / Average electron dose: 20 e/Å2 / Od range: 1.8 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: individual particles
Final angle assignmentDetails: beta gamma
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMAGIC-5 / Details: exact filtered back-projection / Number images used: 31415

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Atomic model buiding 1

Initial model(PDB ID:

2aw7
PDB Unreleased entry

,

2awb
PDB Unreleased entry

)
SoftwareName: O
DetailsProtocol: manual. The domains were separately fitted by manual docking using program O
RefinementProtocol: RIGID BODY FIT
Output model

PDB-2vaz:
Model of the S15-mRNA complex fitted into the cryo-EM map of the 70S entrapment complex.

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