Role of carboxy-terminal region in proofreading function of methionyl-tRNA synthetase in Escherichia coli.
The synthetic and editing functions of three forms of Escherichia coli methionyl-tRNA synthetase with different C-terminal sequences have been compared in vivo and in vitro. These forms include a full-length wild-type dimer (MRS676), a truncated monomer (MRS547) believed to be equivalent to the biologically active large tryptic fragment, and a third form denoted MRS581*. DNA sequencing revealed that MRS581* is predicted to contain 18 additional amino acids from the wild-type full-length sequence at the carboxy terminus of truncated form MRS547, and this is then fused to an additional 16 amino acids encoded by vector pBR322. Both MRS676 and MRS581* were found to edit endogenous homocysteine about 20-fold more efficiently than MRS547 in vivo. However, the three methionyl-tRNA synthetases edited exogenously supplied homocysteine in bacterial cultures to similar extents. Purified proteins exhibited no significant differences in editing function in vitro. Synthetic activity of purified MRS676 in vitro was found to be about 2.5-fold higher per subunit compared to the shorter forms of the enzyme. The C-terminal region in E. coli methionyl-tRNA synthetase is thus suggested to play an important role in editing in vivo, most likely by allowing interaction of the enzyme with the methionine biosynthetic pathway. These data support a model of channeling of at least some metabolites in bacterial protein synthesis.
Gao
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W
Department of Microbiology & Molecular Genetics, Graduate School of Biomedical Sciences, Newark, New Jersey.
Goldman
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E
Jakubowski
H
H
D004926:Escherichia coli / Q000201:enzymology*; D014644:Genetic Variation; D006710:Homocysteine / Q000378:metabolism*; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D006710:Homocysteine; D008718:Methionine-tRNA Ligase