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Aminoacyl-tRNA synthetases perform a critical step in translation by aminoacylating tRNAs with their cognate amino acids. Although high fidelity of aminoacyl-tRNA synthetases is often thought to be essential for cell biology, recent studies indicate that cells tolerate and may even benefit from tRNA misacylation under certain conditions. For example, mammalian cells selectively induce mismethionylation of nonmethionyl tRNAs, and this type of misacylation contributes to a cell's response to oxidative stress. However, the enzyme responsible for tRNA mismethionylation and the mechanism by which specific tRNAs are mismethionylated have not been elucidated. Here we show by tRNA microarrays and filter retention that the methionyl-tRNA synthetase enzyme from Escherichia coli (EcMRS) is sufficient to mismethionylate two tRNA species, tRNAArgCCU and tRNAThrCGU, indicating that tRNA mismethionylation is also present in the bacterial domain of life. We demonstrate that the anticodon nucleotides of these misacylated tRNAs play a critical role in conferring mismethionylation identity. We also show that a certain low level of mismethionylation is maintained for these tRNAs, suggesting that mismethionylation levels may have evolved to confer benefits to the cell while still preserving sufficient translational fidelity to ensure cell viability. EcMRS mutants show distinct effects on mismethionylation, indicating that many regions in this synthetase enzyme influence mismethionylation. Our results show that tRNA mismethionylation can be carried out by a single protein enzyme, mismethionylation also requires identity elements in the tRNA, and EcMRS has a defined structure-function relationship for tRNA mismethionylation. [ABSTRACT FROM AUTHOR]