Two proofreading steps amplify the accuracy of genetic code translation
Ka-Weng Ieong a, Ülkü Uzun a,1, Maria Selmer a, and Måns Ehrenberg a,2
Author Affiliations
a Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden
Edited by Ada Yonath, Weizmann Institute of Science, Rehovot, Israel, and approved October 12, 2016 (received for review July 4, 2016)
Significance
We have discovered that two proofreading steps amplify the accuracy of genetic code reading, not one step, as hitherto believed. We have characterized the molecular basis of each one of these steps, paving the way for structural analysis in conjunction with structure-based standard free energy computations. Our work highlights the essential role of elongation factor Tu for accurate genetic code translation in both initial codon selection and proofreading. Our results have implications for the evolution of efficient and accurate genetic code reading through multistep proofreading, which attenuates the otherwise harmful effects of the obligatory tradeoff between efficiency and accuracy in substrate selection by enzymes.
Abstract
Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-tRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.
ribosome error correction fidelity EF-Tu ternary complex
Footnotes
1Present address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
2To whom correspondence should be addressed. Email: ehrenberg@xray.bmc.uu.se.
Author contributions: K.-W.I. and M.E. designed research; K.-W.I. and Ü.U. performed research; M.S. performed structural analysis; K.-W.I. and M.E. analyzed data; and K.-W.I. and M.E. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1610917113/-/DCSupplemental.
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