Two proofreading steps amplify the accuracy of genetic code translation
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. 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.
Proceedings of the National Academy of Sciences