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Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
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.
2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 1, 131-136 p.Article in journal (Refereed) Published
Abstract [en]

Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.

Place, publisher, year, edition, pages
2012. Vol. 109, no 1, 131-136 p.
Keyword [en]
fidelity, rate-accuracy trade-off, ribosome, protein synthesis, elongation
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-167161DOI: 10.1073/pnas.1116480109ISI: 000298876500031OAI: oai:DiVA.org:uu-167161DiVA: diva2:487314
Available from: 2012-01-31 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Rate and Accuracy of Bacterial Protein Synthesis
Open this publication in new window or tab >>Rate and Accuracy of Bacterial Protein Synthesis
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates.

Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell.

To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 910
Keyword
protein synthesis, ribosome, peptidyl transfer, rate-accuracy trade-off, kinetics
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-171040 (URN)978-91-554-8309-8 (ISBN)
Public defence
2012-05-04, B41, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2012-04-13 Created: 2012-03-15 Last updated: 2012-04-19
2. Accuracy of mRNA Translation in Bacterial Protein Synthesis
Open this publication in new window or tab >>Accuracy of mRNA Translation in Bacterial Protein Synthesis
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch).

Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides. 

We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1306
Keyword
protein synthesis, genetic code, misreading, error hot spots, kinetics, aminoglycoside
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-262901 (URN)978-91-554-9383-7 (ISBN)
Public defence
2015-12-04, B10:2, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-11-13 Created: 2015-09-22 Last updated: 2016-01-13

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Johansson, MagnusZhang, JingjiEhrenberg, Måns

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