Abstract
Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNA in a two-step reaction starting with amino acid activation followed by aminoacyl group transfer to tRNA. To clear mistakes that occasionally occur, some of these enzymes carry out editing activities, acting on the misactivated amino acid (pretransfer editing) or after the transfer on the tRNA (post-transfer editing). The post-transfer editing pathway of leucyl-tRNA synthetase has been extensively studied by structural and biochemical approaches. Here, we report the finding of a tRNA-independent pretransfer editing pathway in leucyl-tRNA synthetases from Aquifex aeolicus. Using a CP1-mutant defective in its post-transfer editing function, we showed that this new editing pathway is distinct from the post-transfer editing site and may occur at the synthetic catalytic site, as recently proposed for other aminoacyl-tRNA synthetases.
Highlights
Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase develop a separated editing domain that is appended to the aminoacylation active site to distinguish structurally similar Leu, Ile, and Val and to eliminate misacylated products [7,8,9]
Fluorescence-based assays and mutational analysis, together with x-ray crystallography studies showing that both pre- and post-transfer editing substrate analogs bind in overlapping sites in the CP1 domain of LeuRS and IleRS, have led to the proposal that misactivated amino acids are translocated from the catalytic site to the editing domain in a tRNA-dependent manner [8, 17,18,19]
It is well accepted that the pretransfer editing of IleRS, valyl-tRNA synthetase, and LeuRS is tRNA-dependent, consistent with the hypothesis that tRNA plays a role in the translocation of AA-AMP from the catalytic
Summary
Preparation of Enzymes and RNA Substrates—AaLeuRS and its mutant D373A were overproduced in Escherichia coli as Histagged proteins and purified by Ni2ϩ-nitrilotriacetic acid chromatography, as previously described [24]. Because Leu-AMP is the cognate product, its binding to LeuRS is very tight, and we could not detect it by the TLC procedure described above without using a modified quenching buffer containing 0.5% SDS, 250 mM ATP, 25 mM EDTA, 200 mM NaAc, pH 4.7. Under these denaturing conditions, the released Leu-AMP could be detected (Fig. S2). The rate constants for the nonenzymatic hydrolysis reactions were obtained by plotting the concentration of Nva-[32P]AMP against time and fitting the data to a first-order decay curve
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