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A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
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2014 (engelsk)Inngår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, nr 5, s. 632-643Artikkel i tidsskrift (Fagfellevurdert) Published
sted, utgiver, år, opplag, sider
2014. Vol. 20, nr 5, s. 632-643
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Identifikatorer
URN: urn:nbn:se:uu:diva-218734DOI: 10.1261/rna.042234.113ISI: 000334677800005OAI: oai:DiVA.org:uu-218734DiVA, id: diva2:696813
Tilgjengelig fra: 2014-02-16 Laget: 2014-02-16 Sist oppdatert: 2017-12-06bibliografisk kontrollert
Inngår i avhandling
1. Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
Åpne denne publikasjonen i ny fane eller vindu >>Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2014. s. 54
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1203
Emneord
Ribosome, protein synthesis, translation, efficiency-accuracy trade-off, kinetics, unnatural amino acids
HSV kategori
Forskningsprogram
Molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-235534 (URN)978-91-554-9103-1 (ISBN)
Disputas
2014-12-17, B41, Uppsala biomedical center (BMC), Uppsala, 13:30 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2014-11-25 Laget: 2014-11-05 Sist oppdatert: 2015-02-03

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