ASN-TRANSFER-RNA IN LACTOBACILLUS-BULGARICUS IS FORMED BY ASPARAGINYLATION OF TRANSFER-RNA AND NOT BY TRANSAMIDATION OF ASP-TRANSFER-RNA

In many organisms (e.g., gram-positive eubacteria) Gln-tRNA is not for med by direct glutaminylation of tRNA(Gln) but by a specific transamid ation of Glu-tRNA(Gln), We wondered whether a similar transamidation p athway also operates in the formation of Asn-tRNA in these organisms, Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferas e of [C-14]Asp-tRNA to [C-14]Asn-tRNA. As no transamidation was observ ed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosom e) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of th e two ORFs (432 amino acids) were the same sand 41% identical with E.c oli AsnRS. When one of the ORFs was expressed in E.coli, it complement ed the temperature sensitivity of an E.coli asnS mutant, S-100 prepara tions of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine, Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS, Thus, the transamidation pathway operates only for Gln-tRNA(Gln) formation in this organism, and possibly in all gram-po sitive eubacteria.