engleski

tRNA-independent proofreading by class I aminoacyl- tRNA synthetases

Isoleucyl- and valyl-tRNA synthetases (IleRS and ValRS, respectively) are well studied class I aminoacyl-tRNA synthetases known to efficiently misactivate noncognate valine and threonine, respectively. In order to keep pace with the tolerable error in protein biosynthesis, they have evolved hydrolytic editing or proofreading to correct these errors. It is well established that correction of misacylated tRNA (post-transfer editing) occurs in a second, spatially separated, active site located at the editing domain. Unfortunately, hydrolysis of noncognate aminoacyl- adenylate (aa-AMP, pre-transfer editing) is more difficult to study. In most class I systems the pre-transfer editing step appeared to require active, aminoacylable tRNA as a cofactor. When active tRNA is present in the assay, transfer and subsequent post-transfer editing also occur, making isolation of the pre-transfer step impossible. Moreover, aminoacyl-adenylates are inherently unstable in solutions which makes their study difficult in vitro. As a consequence, the exact place, mechanism and role for tRNA in noncognate aminoacyl-adenylate hydrolysis is still not understood in detail and several new approaches are undertaken to resolve the current debate in the field [1]. Here we show that both IleRS and ValRS from Escherichia coli possess tRNA-independent pre-transfer editing comprising about 5% of proofreading activity obtained in the presence of cognate tRNA. Our findings do not support the broadly accepted model [2] which assumes that tRNA is indispensable for noncognate aa-AMP hydrolysis. In order to locate whether tRNA-independent pre-transfer editing may occur within the confines of the synthetic site, we have produced several IleRS and ValRS mutants, bearing mutations in the distant editing site. Obtained mutants possess unimpaired activity in cognate aminoacylation and build-up noncognate aminoacyl- tRNA product. Comparison of the steady-state parameters between mutant and wild type enzymes shows that the distant editing site is not involved in tRNA-independent hydrolytic proofreading.

pre-transfer editing; isoleucyl-tRNA synthetase; valyl-tRNA synthetase; hydrolytic proofreading

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