title: CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function.
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.
authors.affiliation: State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, 320 Yue-Yang Road, Shanghai 200031, China.
authors.forename: J F
authors.initials: JF
authors.lastname: Chen
authors.forename: N N
authors.initials: NN
authors.lastname: Guo
authors.forename: T
authors.initials: T
authors.lastname: Li
authors.forename: E D
authors.initials: ED
authors.lastname: Wang
authors.forename: Y L
authors.initials: YL
authors.lastname: Wang
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D016254:Mutagenesis, Insertional; D017393:RNA Editing / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis*
chemical_list: D012346:RNA, Transfer, Amino Acyl; D007532:Isoleucine; D008715:Methionine; D000604:Amino Acyl-tRNA Synthetases