A Non‐Discriminating Aspartyl‐tRNA Synthetase from Halobacterium salinarum

Abstract

The tRNA‐dependent transamidation pathway is the essential route for Asn‐tRNAAsnformation in organisms that lack an asparaginyl‐tRNA synthetase. This pathway relies on anon‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS encoded by aspS), an enzyme withrelaxed tRNA specificity, to form Asp‐tRNAAsn. The misacylated tRNA is then converted toAsn‐tRNAAsn by the action of an Asp‐tRNAAsn amidotransferase. Here we show that AsntRNAAsnformation in the extreme halophile Halobacterium salinarum also occurs by thistransamidation mechanism, and we explore the property of the haloarchaeal AspRS toaspartylate tRNAAsn in vivo and in vitro. Transformation of the E. coli trpA34 strain with theH. salinarum aspS and tRNAAsn genes led to restoration of tryptophan auxotrophy bymissense suppression of the trpA34 mutant with heterologously in vivo formed Asp‐tRNAAsn.The haloarchaeal AspRS works well at low and high (0.1‐3 M) salt concentrations but it isunable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids(H26 and P84) located in the AspRS anticodon binding domain limit the specificity of thisnon‐discriminating enzyme towards tRNAAsn. Thus, as was observed in an archaealdiscriminating AspRS and a bacterial ND‐AspRS, amino acids in these positions influencethe enzyme’s tRNA selection.