The accuracy of the tRNA aminoacylation, ensured by cognate aminoacyl-tRNA synthetase, is of first importance in all living cells. To understand the mechanism underlying the specific recognition of tRNA^Trp by tryptophanyl-tRNA synthetase (TrpRS) from three biological domains, Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA^Trp, representing the three biological domains, were hyperexpressed in Escherichia coli, and examined regarding their efficiency as substrates for tryptophanylation by recombinant B. subtilis, A . fulgidus, and human TrpRS. ^{Trp Trp Trp Trp}...[ Read more ]

The accuracy of the tRNA aminoacylation, ensured by cognate aminoacyl-tRNA synthetase, is of first importance in all living cells. To understand the mechanism underlying the specific recognition of tRNA^Trp by tryptophanyl-tRNA synthetase (TrpRS) from three biological domains, Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA^Trp, representing the three biological domains, were hyperexpressed in Escherichia coli, and examined regarding their efficiency as substrates for tryptophanylation by recombinant B. subtilis, A . fulgidus, and human TrpRS.

Identity elements in tRNA^Trp contribute to the efficient and specific recognition by cognate TrpRS. To determine the identity elements in tRNA^Trp, tRNA^Trp mutants were constructed by site-directed mutagenesis. In B. subtilis tRNA^Trp, A26 was determined to be a negative identity element and U11-A24, G29-C41, and U31-A39 were determined to be minor identity elements through in vitro tryptophanylation assays. Of these tRNA^Trp mutants, U11C-A24G and A26G displayed significant structural change in the core region of tRNA^Trp, revealed by NMR study. The U11C-A24G also affected the expression level of mutant tRNA. In bovine tRNA^Trp, mutations in either the discriminator base (A73G) or first base pair (G1A-C72U) of acceptor stem of bovine tRNA^Trp, resulted in a drastic loss of tryptophanylation efficiency, suggesting that A73 and G1-C72 were major positive identity elements in the eukaryotic tRNA^Trp. The synergetic effects between these two elements suggested a crucial domain required for effective recognition by human TrpRS. The increase of charging ability for G26A suggests that G26 is a negative identity element in bovine tRNA^Trp. In A . fulgidus tRNA^Trp, A73 also serves as a major positive identity element, which resembles that of eukaryotic tRNA^Trp. Like tRNA^Trp from the other two kingdoms, the G26A mutant of A . fulgidus tRNA^Trp exhibited increased activity. Therefore, N26 exerts a large influence on tRNA^Trp tryptophanylation and serves as a negative identity element towards cognate TrpRS.

Since the discriminator base (N73) is a major identity element in tRNA^Trp, the recognition patterns of N73 by TrpRS from the three kingdoms were compared. Results revealed two distinct preference profiles regarding the N73 of tRNA^Trp substrates: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS based on the k_cat/K_M values. The preference for G73 in tRNA^Trp by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA^Trp from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria.