Two Distinct Routes for Asparaginyl‐tRNA Formation in B. halodurans

Abstract

In nature, two distinct routes have evolved for attaching the amino acid asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct pathway Asn is ligated to tRNAAsn by an asparaginyl-tRNA synthetase (AsnRS). In a second, two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) first misacylates Asp to tRNAAsn. This tRNA-bound Asp is then amidated to Asn by the amidotransferase GatCAB. Bioinformatic analysis shows that the Gram-positive Bacillus halodurans can use the direct route. However, whether it uses the indirect route depends on whether its AspRS is non-discriminating. Using both in vivo and in vitro assays, we demonstrate the B. halodurans AspRS can aspartylate tRNAAsn and can be used with GatCAB to form Asn-tRNAAsn. Aminoacylation of tRNAAsn by AsnRS is five-fold more efficient than with AspRS. Retention of the two-step pathway for Asn-tRNA formation in B. halodurans may be vestigial though is might be advantageous unde...