engleski

Determinants for tRNA-dependent pre-transfer editing in the synthetic site of isoleucyl-tRNA synthetase

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 non-cognate 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 pre-transfer editing to hydrolyze non-cognate valyl-adenylate prior to aminoacyl-tRNA formation. Based on 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 pre-transfer 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 pre-transfer editing accounts for nearly one- third of 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 pre-transfer editing as the predominant proofreading pathway, since 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.

isoleucyl-tRNA synthetase ; pre-transfer editing ; proofreading ; active site mutants

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