The discriminator bases G 73 in human tRNAser and A73 in tRNALeu have significantly different roles in the recognition of aminoacyl-tRNA synthetases

Abstract

The recognition of human tRNALeu or tRNASer by cognate aminoacyl-tRNA synthetases has distinct requirements. Only one base change (A73→G) in tRNALeu is required to generate an efficient serine acceptor in vitro, whereas several changes in three structural domains (the acceptor stem, DHU loop and long extra arm) of tRNASer are necessary in order to produce a leucine acceptor. Hence, the molecular basis for the discrimination between human tRNASer and tRNALeu by the seryl-tRNA synthetase depends almost exclusively on a highly specific recognition of the discriminator base G 73. In order to elucidate the specific role of the functional groups of this base in discrimination, tRNASer constructs were made which contain the artificial base analogues 2-aminopurine riboside or inosine at the discriminator position 73. Aminoacylation of these constructs by a HeLa S100 extract showed that molecules with 2-aminopurine riboside, but not with inosine, in position 73 could be serylated at low efficiency. However, the 2-aminopurine riboside and the inosine derivatives of tRNAser were equally efficient competitive inhibitors of serylation, whereas tRNAsSer with any other natural base at position 73 did not competitively inhibit serylation of tRNASer. This was in contrast to leucylation of tRNALeu, where tRNALeu transcripts with any other nucleotide in the discriminator position acted as strong competitive inhibitors. These results suggest that the discriminator bases in human tRNASer and tRNALeu play completely different roles in recognition of the tRNAs by their cognate aminoacyl-tRNA synthetases.