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Enhanced proofreading attenuates initial selection error hot spots in genetic code translation by transfer RNAs
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A system for cell free protein synthesis with E. coli components of high purity was used in conjunction with fast kinetics quench-flow measurements to characterize the accuracy of peptie bond formation by ribosomes with initiator tRNAfMet in P site and different codons in the A site. We used Glu-tRNAGlu, Lys-tRNALys and Phe-tRNAPhe in ternary complexes with EF-Tu and GTP to select ribosomes programmed with their respective cognate codons in competition with ribosomes containing near-cognate codons. Variation of the free Mg2+ concentration in the in vitro buffer system was used to calibrate its accuracy to that of codon selection by Glu-tRNAGlu in living E. coli cells, previously estimated from the residual activity of a beta-galactosidase mutant in which the codon for an essential Glu had been altered to near cognate codons. At 2.3 mM free Mg2+ concentration, the accuracy in the living cell agreed with that in the test tube, a feature making our biochemistry directly relevant to bacterial physiology. We found that the total accuracy of tRNA selection varied by five orders of magnitude depending on the type of tRNA, type of mismatch and mismatched codon position. We partitioned the total accuracy into initial selection of ternary complex before GTP hydrolysis on EF-Tu and proofreading selection of aminoacyl-tRNA after GTP hydrolysis. We found the contribution of proofreading to be strongly positively correlated with the accuracy of initial selection in its high range. As initial selection decreased further the proofreading contribution to accuracy increased, rather than decreased, a feature neutralizing potentially disastrous missense error hot spots associated with, in particular, tRNAGlu.    

Keyword [en]
protein synthesis, genetic code, misreading, error hot spots, kinetics, initial selection, proofreading, total accuracy
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-264957OAI: oai:DiVA.org:uu-264957DiVA: diva2:862232
Available from: 2015-10-20 Created: 2015-10-20 Last updated: 2016-01-13
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1306
protein synthesis, genetic code, misreading, error hot spots, kinetics, aminoglycoside
National Category
Biochemistry and Molecular Biology
Research subject
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)
Available from: 2015-11-13 Created: 2015-09-22 Last updated: 2016-01-13

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