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Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

ABSTRACT: Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA?? end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA?? accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A?? is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C?? and C?? selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA?? binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA?? homogeneity of tRNAs.

SUBMITTER: Zhou XL

PROVIDER: S-EPMC3203616 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

Zhou Xiao-Long XL Du Dao-Hai DH Tan Min M Lei Hui-Yan HY Ruan Liang-Liang LL Eriani Gilbert G Wang En-Duo ED

Nucleic acids research 20110720 20

Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA₇₆ end to ...[more]

PMID: 21775341

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Project description:UnlabelledThe antibiotic streptothricin (ST) possesses an amino sugar bound to an l-β-lysine (β-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a β-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics.ImportanceThe antibiotic streptothricin (ST) possesses an amino sugar bound to an l-β-lysine (β-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than β-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

Dataset Information

Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

Publications

Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

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