Determinants for tRNA-Dependent Pretransfer Editing in the Synthetic Site of Isoleucyl-tRNA Synthetase
| Autor: | Ita Gruić-Sovulj, John J. Perona, Morana Dulic |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Isoleucine-tRNA Ligase
Models Molecular Isoleucine—tRNA ligase Molecular Sequence Data Aminoacylation Biology Biochemistry Article RNA Transfer Escherichia coli Amino Acid Sequence Binding site Transfer RNA Aminoacylation RNA Transfer Ile isoleucyl-tRNA synthetase pre-transfer editing proofreading active site mutants chemistry.chemical_classification Binding Sites Sequence Homology Amino Acid Escherichia coli Proteins Hydrolysis Translation (biology) Biosynthetic Pathways Protein Structure Tertiary Amino acid Kinetics chemistry RNA editing Transfer RNA Tyrosine RNA Editing Protein Binding |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi5007699 |
| Popis: | The accurate expression of genetic information relies on the fidelity of amino acid-tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs. |
| Databáze: | OpenAIRE |
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