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Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 43, 17955-17962 p.Article in journal (Refereed) Published
Abstract [en]

Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA Phe. The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA Phe derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA Phe. We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.

Place, publisher, year, edition, pages
2012. Vol. 134, no 43, 17955-17962 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-186030DOI: 10.1021/ja3063524ISI: 000310483500024OAI: oai:DiVA.org:uu-186030DiVA: diva2:572554
Available from: 2012-11-28 Created: 2012-11-27 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
Open this publication in new window or tab >>Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids?

Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition.

By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches.

We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis.

Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1203
Keyword
Ribosome, protein synthesis, translation, efficiency-accuracy trade-off, kinetics, unnatural amino acids
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-235534 (URN)978-91-554-9103-1 (ISBN)
Public defence
2014-12-17, B41, Uppsala biomedical center (BMC), Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2014-11-25 Created: 2014-11-05 Last updated: 2015-02-03

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Ieong, Ka-WengPavlov, Michael Y.Kwiatkowski, MarekForster, Anthony C.Ehrenberg, Måns

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