Dual Routes for Bacillus halodurans Asparaginyl‐tRNA Formation

Abstract

Two evolutionary distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis, are found in nature. In the first, asparaginyl‐tRNA synthetase directly ligates Asn to tRNAAsn in an ATP‐dependent manner. In the second pathway, a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS) first ligates Asp to tRNAAsn and GatCAB then modifies the tRNA‐bound Asp to Asn by transamidating the amino acid. Our bioinformatics analysis of the Gram‐positive Bacillus halodurans suggests it may encode the indirect pathway in addition to the direct one. Encoding the indirect Asn‐tRNAAsn biosynthetic pathway requires an ND‐AspRS. We are testing whether the B. halodurans AspRS is non‐discriminating (i.e., uses tRNAAsn as a substrate along with tRNAAsp). Using the E. coli JF448 system, we have determined the B. halodurans AspRS is non‐discriminating and works in concert with GatCAB to synthesize Asn in a tRNA‐dependent manner. Further verification of the nature of the AspRS using the E. coli TrpA34 system and in vitro studies are underway. We speculate retention of both pathways for Asn‐tRNAAsn synthesis in B. halodurans may be vestigial and that a number of other bacteria may also maintain both routes.