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Research Paper
A Non‐Discriminating Aspartyl‐tRNA Synthetase from Halobacterium salinarum
Alexander Machado Cardoso, Carla Polycarpo, Orlando Bonifácio Martins and Dieter Söll
volume 3 | issue 3
july/august/september 2006Pages: 110 - 114
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The tRNA‐dependent transamidation pathway is the essential route for Asn‐tRNAAsn formation in organisms that lack an asparaginyl‐tRNA synthetase. This pathway relies on a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp‐tRNAAsn. The misacylated tRNA is then converted to Asn‐tRNAAsn by the action of an Asp‐tRNAAsn amidotransferase. Here we show that AsntRNAAsn formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNAAsn in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNAAsn genes led to restoration of tryptophan auxotrophy by missense 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 is unable 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 this non‐discriminating enzyme towards tRNAAsn. Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND‐AspRS, amino acids in these positions influence the enzyme’s tRNA selection.
We now provide open access to journal articles published online for one year or more. This article may be downloaded at the following link:
Download PDF If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.