Three G·C Base Pairs Required for the Efficient Aminoacylation of tRNATrp by Tryptophanyl-tRNA Synthetase from Bacillus subtilis

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Abstract
Acceptor stem is an essential region in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. In this study, a library containing 20 nt random region and tryptophanyl-tRNA synthetase (TrpRS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the tRNA(Trp) acceptor stem sequence that is required for B. subtilis TrpRS recognition. After three rounds of selection, the TrpRS binding RNAs dominate the RNA pool. The aptamers share a common structure of three G.C base pairs, which was also found in the acceptor stem of wild-type B. subtilis tRNA(Trp). A series of tRNA(Trp) variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (k(cat)/K(M)) were measured with the aid of TrpRS from B. subtilis. The mutants that possess the three G.C base pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B. subtilis tRNA(Trp), while the G73 discriminator base itself cannot confer efficient aminoacylation to the tRNA(Trp) molecule. Thus, these three base pairs (G2.C71, G3.C70, and G4.C69) in the B. subtilis tRNA(Trp) acceptor stem were established to be new identity elements, and their importance was between the previously characterized major element G73 and minor elements A1/U72 and G5/C68. The minimum set of identity elements that is required to confer efficient aminoacylation by B. subtilis TrpRS included G73, G2.C71, G3.C70, and G4.C69.