||In hope of further understanding the evolution of tRNAs, mutational analysis of B.subtilis tRNA for Trp was carried out. In the study, bases of B.subtiZis tRNATrp which are conserved among prokaryotes but not conserved with the eukaryotes were mutagenized. The mutant tRNAs U11C-A24G, A26G, U31A-A39U and G29U-C41A were obtained through in vivo heterologous expression in Escherichia coli. The Trp-acceptance activities of the mutant B.subtilis tRNATrp were compared with that of the wild-type molecule. The results show that the mutant tRNATrps U11C-A24G, A26G and U31A-A39U are lower in reactivity toward its cognate TrpRS than the wild-type. The measurements suggest that, in addition to the major identity element G73 and the minor identity elements A1-U72, G5-C68 and A9 previously identified by our laboratory, the residues U11-A24 act as another major identity element and A26, U31-A39 as minor identity elements. To further characterize this phylogenetic study of tRNATrp, the expression of human tryptophanyl-tRNA synthetase was carried out. The gene which encodes the human tryptophanyl-tRNA synthetase has been subcloned into an expression vector, pTrcHisB and expressed in E.coli. As the prokaryotic system of codon usage is different from that of the eukaryotes, silent mutations have been performed in order to achieve a higher level of human TrpRS expression in E.coli. The purified human TrpRS was used to test for the charging activities of B.subtilis tRNATrp mutants. The study on the mutant tRNATrp U11C-A24C illustrates that the major identity element U11-A24 is not involved in the recognition. Differential scanning calorimetric study on this base pair suggests its main function is the maintenance the correct tertiary structure for TrpRS recognition. The results also show that both G73A and G73C mutants are susceptible to tryptophanylation, which is comparable to the strigent requirement of G73 in the prokaryotic system. This may be due to the preference difference between the two enzymes in identifying particular functional groups or structural features for recognition. The study further demonstrates the usefulness of phylogenetic comparison as an experimental approach for defining the identity elements of tRNAs.