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Accuracy of mRNA Translation in Bacterial Protein Synthesis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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 [en]
protein synthesis, genetic code, misreading, error hot spots, kinetics, aminoglycoside
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-262901ISBN: 978-91-554-9383-7 (print)OAI: oai:DiVA.org:uu-262901DiVA: diva2:862028
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
List of papers
1. Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
Open this publication in new window or tab >>Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
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.

Keyword
fidelity, rate-accuracy trade-off, ribosome, protein synthesis, elongation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-167161 (URN)10.1073/pnas.1116480109 (DOI)000298876500031 ()
Available from: 2012-01-31 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
2. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome
Open this publication in new window or tab >>Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome
2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 31, 9602-9607 p.Article in journal (Refereed) Published
Abstract [en]

We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

Keyword
protein synthesis, genetic code, misreading, error hot spots, kinetics
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-261240 (URN)10.1073/pnas.1506823112 (DOI)000358930600052 ()26195797 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-09-07 Created: 2015-08-31 Last updated: 2017-12-04Bibliographically approved
3. Enhanced proofreading attenuates initial selection error hot spots in genetic code translation by transfer RNAs
Open this publication in new window or tab >>Enhanced proofreading attenuates initial selection error hot spots in genetic code translation by transfer RNAs
(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
protein synthesis, genetic code, misreading, error hot spots, kinetics, initial selection, proofreading, total accuracy
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-264957 (URN)
Available from: 2015-10-20 Created: 2015-10-20 Last updated: 2016-01-13

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