Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.

Abstract

Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, twodimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells. Aminoacylation of tRNA is a two-step reaction. In the first step, an amino acid (AA) is activated to form enzyme (E)-bound aminoacyl adenylate.In the second step, the amino acid is transferred from the adenylate to tRNA.E-AA-AMP + tRNAAA -* E + AAAtRNAAA + AMP Extraordinary fidelity of the tRNA aminoacylation reaction is due to enzymatic proofreading or editing activities of aminoacyl-tRNA synthetases (5, 6). It has been proposed that aminoacyl-tRNA synthetases can exercise kinetic proofreading of incorrect substrates (7,8). Editing can occur by the hydrolysis of the noncognate aminoacyl adenylate (9-11) or the aminoacyl-tRNA (12-15). Both editing mechanisms have been directly demonstrated in vitro. Although it has been inferred from the in vitro studies that editing is also important in vivo, direct evidence for this was missing.The aminoacyl-adenylate proofreading pathway originally discovered with valyl-tRNA synthetase (9, 10) has subsequently been established in vitro to be of major importance with several other synthetases (11). In particular, methionyl-, isoleucyl-, and valyl-tRNA synthetases edited misactivated homocysteine by the adenylate pathway with the formation of homocysteine thiolactone. This feature of the homocysteine editing reaction provides a means to assay for editing in vivo by looking for a special product of editing, i.e., homocysteine thiolactone. I now report that homocysteine thiolactone is in fact synthesized in vivo by the bacterium Escherichia coli. The data indicate that the thiolactone synthesis in E. coli is due to efficient in vivo editing of homocysteine by methionyl-tRNA synthetase, thus establishing the existence and importance of proofreading in vivo. MATERIALS AND METHODSBacterial Strains and Plasmids. Wild-type E. coli K-38 was obtained from N. Zinder (Rockefeller University). E. coli K-12 metE was from N. Brot (Roche Institute of Molecular Biology). The following strains were obtained from the E. coli Genetic Stock Center (Yale University): RK4536 (metE metH); CS50 (metG); CA274 (trp49); AB2575 (ilvD); Hfr3000YA73 (thrB). CR147 was from M. Cashel (National In...

Homocysteine Toxicity inEscherichia coliIs Caused by a Perturbation of Branched-Chain Amino Acid Biosynthesis

“…This reaction is inhibited strongly in the presence of methionine, since methionine competes efficiently with Hcy for binding to the enzyme (10). To determine whether the inhibitory effects of Hcy required the formation of homocysteine thiolactone, we performed growth experiments with Hcy in the presence or absence of either 0.2 or 2 mM methionine.…”

“…These results suggest that Hcy may have more complex effects on cell physiology at higher concentrations. Hcy is known to compete with methionine and isoleucine for binding to their respective tRNA synthetases (10,13), and it could be that at higher Hcy concentrations this competition depletes the cell of charged tRNA species, resulting in reduced rates of protein synthesis. Under these conditions homocysteine thiolactone is likely to accumulate in the cytoplasm due to the editing function of the methionyl and isoleucyl tRNA synthetases (13).…”

Is Endothelial Nitric Oxide Synthase a Moonlighting Protein Whose Day Job is Cholesterol Sulfate Synthesis? Implications for Cholesterol Transport, Diabetes and Cardiovascular Disease

“…Mammalian systems are similar to E. coli with respect to the role that methionyl-tRNA synthetase plays in methionine incorporation into proteins [167]. Studies on E. coli have demonstrated that, due to competition of homocysteine with methionine in the process that assembles amino acid chains, one molecule of homocysteine is edited to produce homocysteine thiolactone per 109 molecules of methionine incorporated into protein in vivo [168]. Thus, an excess need for protein synthesis would lead to an excess synthesis of homocysteine thiolactone.…”

Photosynthate costs associated with the utilization of different nitrogen–forms: influence on the carbon balance of plants and shoot–root biomass partitioning

“…It follows that, at this step alone, the synthesis of a polypeptide containing n amino acid residues consumes the equivalent of (2n) ATP. However, an incorrect amino acid and\or tRNA might also be activated or charged (Hopfield, 1974 ;Hopfield et al, 1976 ;Savageau & Freter, 1979 ;Cramer & Freist, 1987 ;Jakubowski, 1995 ;Freist, Sternbach & Cramer, 1996). The accuracy of this step of protein synthesis (amino acid selection by tRNA synthetases) is increased by, inter alia, proofreading\editing pathways whereby the misactivated aminoacyl-adenylate and\or aminoacyl-tRNA complexes are rejected by a mechanism which involves an additional ATP hydrolysis (Hopfield, 1974 ;Hopfield et al, 1976 ;Savageau & Freter, 1979 ;Mulvey & Fersht, 1977 ;Fersht, 1986 ;Jakubowski & Goldman, 1992 ;Freist et al, 1996).…”