engleski

Aminoacyl-tRNA synthetases: accuracy of substrate recognition and catalysis

Aminoacyl-tRNA synthetases (aaRS) are group of enzymes that catalyze attachment of amino acid to cognate tRNA in a two-step reaction. First amino acid is activated in an ATP-dependent manner to yield an aminoacyl-adenylate intermediate and pyrophosphate. In the second step, amino acid is transferred to tRNA producing aminoacyl-tRNA with concomitant release of AMP. The accuracy of this reaction is essential for the fidelity of protein synthesis and hence cell survival. To obtain a high level of specificity in tRNA recognition, synthetases utilize an extensive network of interactions that participate in direct and indirect readout of cognate macromolecular substrate. Components of tRNA's structure that are responsible for the productive interaction with cognate aaRS are known as identity elements. We have studied the way of tRNA recognition and tRNA's identity elements in yeast and methanogenic archaea in order to elucidate whether this fundamental biological process vary between different domains of life. We observed that they do employ different modes of recognition between seryl-tRNA synthetase and cognate tRNASer. Accurate recognition of cognate amino acid is more challenging than recognition of cognate tRNA. Amino acids are small substrates with many of them sharing similar chemical structures of their side chains. As a result, some aaRSs proceed in misactivation of noncognate amino acid and subsequent transfer of amino acid to tRNA (forming misacyl-tRNA). To correct these errors, synthetases have evolved mechanistically complex and energetically expensive hydrolytic editing or proofreading. 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 aminoacyl-adenylate) are still open questions. We have shown previously that aminoacyl-adenylate may be corrected in the hydrolytic site that is spatially adjacent or overlapping with the synthetic active site rather than at the separate domain used for post-transfer editing. Our aim is to elucidate the detailed mechanism and propose a model for pre-transfer editing. Therefore, isoleucyl- and valyl-tRNA synthetases, well known highly editing synthetases, were chosen as molecular models in biochemical and kinetic analyses.

aminoacyl-tRNA synthetases; pre-transfer editing; tRNA identity

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