Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs.

The ribosome uses initial and proofreading selection of aminoacyl-tRNAs for accurate protein synthesis. Anomalously high initial misreading in vitro of near-cognate codons by tRNAHis and tRNAGlu suggested potential error hotspots in protein synthesis, but in vivo data suggested their partial neutralization. To clarify the role of proofreading in this error reduction, we varied the Mg2+ ion concentration to calibrate the total accuracy of our cell-free system to that in the living Escherichia coli cell. We found the total accuracy of tRNA selection in our system to vary by five orders of magnitude depending on tRNA identity, type of mismatch, and mismatched codon position. Proofreading and initial selection were positively correlated at high, but uncorrelated at low initial selection, suggesting hyperactivated proofreading as a means to neutralize potentially disastrous initial selection errors.