PMID:10828991
| Citation |
Chen, JF, Guo, NN, Li, T, Wang, ED and Wang, YL (2000) CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function. Biochemistry 39:6726-31 |
|---|---|
| Abstract |
The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function. |
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| Keywords |
Amino Acyl-tRNA Synthetases/chemistry; Amino Acyl-tRNA Synthetases/genetics; Amino Acyl-tRNA Synthetases/metabolism; Circular Dichroism; Cloning, Molecular; Escherichia coli/enzymology; Isoleucine/metabolism; Kinetics; Methionine/metabolism; Mutagenesis, Insertional; RNA Editing/genetics; RNA, Transfer, Amino Acyl/biosynthesis |
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