Proofreading in vivo: Editing of Homocysteine by Methionyl-tRNA Synthetase in Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-06, Vol.87 (12), p.4504-4508
• Main Author: Jakubowski, Hieronim
• Format: Article
• Language: English
• Subjects: AMINO ACIDS; Amino Acids - metabolism; Amino Acyl-tRNA Synthetases - metabolism; BACTERIA; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOCHEMICAL REACTION KINETICS; Biochemistry; Biological and medical sciences; BIOLOGICAL PATHWAYS; BIOSYNTHESIS; CARBOXYLIC ACIDS; CELL CONSTITUENTS; Centrifugation; CHROMATOGRAPHY; Cultured cells; DAYS LIVING RADIOISOTOPES; ENZYMES; ESCHERICHIA COLI; Escherichia coli - enzymology; Escherichia coli - genetics; ESTERS; EVEN-ODD NUCLEI; Fundamental and applied biological sciences. Psychology; HETEROCYCLIC COMPOUNDS; HOMOCYSTEINE; Homocysteine - analogs & derivatives; Homocysteine - biosynthesis; Homocysteine - isolation & purification; Homocysteine - metabolism; HYDROLASES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LACTONES; LIGHT NUCLEI; Methionine-tRNA Ligase - genetics; Methionine-tRNA Ligase - metabolism; MICROORGANISMS; Molecular and cellular biology; Molecular genetics; Mutation; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; PLASMIDS; Proofreading; Protein synthesis; RADIOISOTOPES; REACTION KINETICS; SEPARATION PROCESSES; Sulfates - metabolism; SULFUR 35; SULFUR ISOTOPES; Sulfur Radioisotopes; SYNTHESIS 550201 > Biochemistry-- Tracer Techniques; THIN-LAYER CHROMATOGRAPHY; TRACER TECHNIQUES; Transfer RNA; Translation. Translation factors. Protein processing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.12.4504

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, two-dimensional TLC analysis of35S-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.