Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli.

Previous in vitro work has shown that Escherichia coli methionyl-tRNA synthetase has a limited ability to discriminate against cognate methionine in the editing site designed for noncognate homocysteine. As a result, a small fraction of the correct product Met-tRNA is deacylated with the formation of a cyclic sulfonium compound, S-methyl-homocysteine thiolactone. This is exploited here to estimate energy costs associated with the destruction of a correct product by methionyl-tRNA synthetase in bacterial cells. In vivo measurements of S-methyl-homocysteine thiolactone indicate that in Escherichia coli 3.3 molecules of Met-tRNA are destroyed by deacylation per 10,000 molecules of Met-tRNA successfully transferring methionine to protein. This number of destroyed molecules of a correct product, Met-tRNA, is 30 times lower than the number of destroyed molecules of an incorrect product, homocysteinyl adenylate. Thus, most of the energy cost of proofreading in vivo is due to editing of the noncognate amino acid.