tRNA selection and kinetic proofreading in translation
Abstract
Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.
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Tetracycline sterically blocks aa-tRNA entry, resulting in cognate and near-cognate inhibition of initial selection between CR and GA states.
“By contrast, tetracycline sterically blocks aa-tRNA entry, resulting in cognate and near-cognate inhibition of initial selection between CR and GA states (15, 16).”
From Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors (2016) · cites this paper as [15]
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- Authors
- Blanchard, S. C., Gonzalez, R. L., Kim, H. D., Chu, S., & Puglisi, J. D.
- Affiliations
- Stanford University (US)
- Stanford Medicine (US)
- Corresponding author
- Chu, Steven
- Puglisi, Joseph D
- Venue
- Nature Structural & Molecular Biology
- Published
- 2004-09-26
- Volume
- 11
- Issue
- 10
- Pages
- 1008-1014
- ISSN
- 1545-9985 (ISSN-L)
- 1545-9993
- OpenAlex
- W2124081058
- Cited by
- 466
- References
- 47
- Citations by year
- 2026: 6 · 2025: 6 · 2024: 16 · 2023: 10 · 2022: 18 · 2021: 11 · 2020: 16 · 2019: 13 · 2018: 20 · 2017: 21 · 2016: 27 · 2015: 18 · 2014: 22 · 2013: 33 · 2012: 26
- FWCI
- 7.82
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Structural & Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM051266, GM51266)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- tRNA selection, kinetic proofreading, translation initiation, ribosome, aminoacyl-tRNA, codon-anticodon recognition, sarcin-ricin loop, tetracycline, nonhydrolyzable GTP analogs, single-molecule FRET, ribosome recycling
- MeSH terms
- Codon, Energy Transfer, Fluorescence, RNA, Transfer, Protein Biosynthesis, GTP Phosphohydrolases
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