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Aminoacyl-tRNA synthetases (aaRS) catalyze formation of the cognate pair between amino acid and tRNA via ATP-dependent two-step reaction. Firstly amino acid is activated to yield an aminoacyl-adenylate (aa-AMP) intermediate followed by the transfer of amino acid to tRNA. The accuracy of this reaction is essential for the fidelity of protein biosynthesis since attached amino acid is not additionally proofread during the selection of aminoacylated tRNAs at the ribosome. While aaRSs are able to recognize cognate tRNA substrate with high accuracy due to large contact surface area, accurate selection of cognate amino acid from near-cognate ones could be critical because of smaller contact area and similar chemical structures of their side chains. As a result, some aaRSs proceed in misactivation of noncognate amino acid and its subsequent transfer to tRNA. In order to keep pace with the tolerable error in protein biosynthesis, aaRSs 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. However, the exact place, mechanism and role for tRNA in pre-transfer editing (hydrolysis of noncognate aa-AMP) are still not understood in detail and several new lines of evidence [1] question the model previously proposed [2]. Here we show that both isoleucyl and valyl-tRNA-synthetases (IleRS and ValRS, respectively) possess tRNA-independent pre-transfer editing comprising 5% of overall proofreading activity. Addition of tRNA highly stimulates proofreading but the mechanism is not completely understood yet. Our findings contradict the broadly accepted model [2] which assumes that tRNA is indispensable for noncognate aa-AMP hydrolysis. Several IleRS and ValRS mutants, bearing mutations in the distant editing site, were cloned and purified. Steady-state and pre-steady state analyses are performed at the moment to clarify whether this activity occurs within the confines of the synthetic site and to elucidate the mechanism of hydrolysis in the absence of tRNA. In order to prepare huge amount of tRNA for kinetic analysis of tRNA-dependent proofreading, tRNAIle and tRNAVal major isoacceptor genes were cloned and overexpressed in vivo. Up to now, tRNAIle isoacceptor has been isolated by reverse-phase chromatography. [1] S.S. Yadavalli, K. Musier-Forsyth and M. Ibba, Proc. Natl. Acad. Sci. USA, 105 (2008) 19031 [2] A.C. Bishop, T.K. Nomanbhoy and P. Schimmel, Proc. Natl. Acad. Sci. USA, 99, (2002) 585 |
