Two proofreading steps amplify the accuracy of genetic code translation.
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.
Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden.
Ka-Weng
0000-0003-4518-0618
KW
Ieong
Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden.
Ülkü
Ü
Uzun
Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden.
Maria
M
Selmer
Department of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden ehrenberg@xray.bmc.uu.se.
Måns
M
Ehrenberg
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D005815:Genetic Code / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*; D050801:Ternary Complex Factors / Q000235:genetics
D012333:RNA, Messenger; D050801:Ternary Complex Factors; D006153:Guanosine Diphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases
EF-Tu; error correction; fidelity; ribosome; ternary complex