Recognition by Tryptophanyl-tRNA Synthetases of Discriminator Base on tRNATrp from Three Biological Domains

Qing Guo,Qingguo Gong,Ka-Lok Tong,Bente Vestergaard,Annie Costa,Jean Desgres,Mansim Wong,Henri Grosjean,Guang Zhu,J Tze-Fei Wong,Hong Xue

doi:10.1074/jbc.m111745200

Abstract

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. 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. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.

Highlights

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNATrp discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNATrp, representing the three biological domains
There were only four identical nucleotides between B. subtilis and bovine tRNATrp unshared by A. fulgidus tRNATrp. These results suggest that, sequence divergence is large between bovine and B. subtilis tRNATrp, A. fulgidus tRNATrp occupies an intermediate phylogenetic position between them with unmistakable resemblance to both of them
The bulk of bovine or A. fulgidus tRNATrp was eluted in one major HPLC peak as revealed by dot-blot hybridization (Fig. 2) in contrast to the multiple HPLC peaks observed with B. subtilis tRNATrp differing in nucleotide modifications [15]

Summary

Introduction

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNATrp discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNATrp, representing the three biological domains. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair caused reduction of Trp-tRNATrp formation These observations identify a tRNATrp structural region near the end of acceptor stem comprising. To gain further insight into this evolutionary change in N73 recognition, in this study the interactions between TrpRS and tRNATrp from all three biological domains of bacteria, archaea, and eukarya were compared. A complete set of Bacillus subtilis, A. fulgidus, and bovine tRNATrp carrying different base substitutions at N73 was hyperexpressed in Escherichia coli and examined in terms of their efficiency as substrates for tryptophanylation by B. subtilis, A. fulgidus, and human TrpRS. To determine any structural role of N73 in bringing about tryptophanylation, solution NMR spectroscopy was employed to compare the structures of wild-type A73 bovine tRNATrp and its G73 mutant, which were respectively, the best and the poorest substrates among the bovine tRNATrp N73 variants toward reaction with human TrpRS

Methods

Results

Conclusion