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- PDB-7uri: allo-tRNAUTu1A in the A site of the E. coli ribosome -

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Basic information

Entry
Database: PDB / ID: 7uri
Titleallo-tRNAUTu1A in the A site of the E. coli ribosome
ComponentsAllo-tRNAUTu1A
KeywordsRNA / tRNA / selenocysteine
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesmetagenome (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsZhang, J. / Prabhakar, A. / Krahn, N. / Vargas-Rodriguez, O. / Krupkin, M. / Fu, Z. / Acosta-Reyes, F.J. / Ge, X. / Choi, J. / Crnkovic, A. ...Zhang, J. / Prabhakar, A. / Krahn, N. / Vargas-Rodriguez, O. / Krupkin, M. / Fu, Z. / Acosta-Reyes, F.J. / Ge, X. / Choi, J. / Crnkovic, A. / Ehrenberg, M. / Viani Puglisi, E. / Soll, D. / Puglisi, J.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122560 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122560-05S1 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM51266 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI15046 United States
Department of Energy (DOE, United States)DE-FG0298ER2031 United States
Cystic Fibrosis FoundationPUGLIS20G0 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Uncovering translation roadblocks during the development of a synthetic tRNA.
Authors: Arjun Prabhakar / Natalie Krahn / Jingji Zhang / Oscar Vargas-Rodriguez / Miri Krupkin / Ziao Fu / Francisco J Acosta-Reyes / Xueliang Ge / Junhong Choi / Ana Crnković / Måns Ehrenberg / ...Authors: Arjun Prabhakar / Natalie Krahn / Jingji Zhang / Oscar Vargas-Rodriguez / Miri Krupkin / Ziao Fu / Francisco J Acosta-Reyes / Xueliang Ge / Junhong Choi / Ana Crnković / Måns Ehrenberg / Elisabetta Viani Puglisi / Dieter Söll / Joseph Puglisi /
Abstract: Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ...Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
History
DepositionApr 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
y: Allo-tRNAUTu1A


Theoretical massNumber of molelcules
Total (without water)28,7821
Polymers28,7821
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain Allo-tRNAUTu1A


Mass: 28782.008 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Oil polluted marine microbial communities from Coal Oil Point, Santa Barbara, California, USA - Santa Barbara Oil Seep Sample 6 (Crude oil metagenome 6, ASSEMBLY_DATE=20131204)
Source: (synth.) metagenome (others)

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: E. coli 70S ribosome with allo-tRNAUTu1A in the A site
Type: RIBOSOME / Entity ID: all / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: CTFFIND / Version: 4 / Category: CTF correction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 10850093
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 424828 / Symmetry type: POINT

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