Three GC Base Pairs Required for the Efficient Aminoacylation of tRna Trp by Tryptophanyl-tRna Synthetase from Bacillus subtilis

Xu, F.; Jiang, G.; Li, W.; He, X.; Jin, Y.; Wang, D.

Biochemistry 41(25): 8087-8092

2002


ISSN/ISBN: 0006-2960
DOI: 10.1021/bi015881g
Accession: 067188462

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Abstract
Acceptor stem is an essential region in the recognition of t RNAs by their cognate aminoacyl-t Rna synthetase. In this study, a library containing 20 nt random region and tryptophanyl-t Rna synthetase (Trp RS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the t RNATrp acceptor stem sequence that is required for B. subtilis Trp Rs recognition. After three rounds of selection, the Trp Rs 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 t RNATrp. A series of t RNATrp variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (kcat/KM) were measured with the aid of Trp Rs 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 t RNATrp, while the G73 discriminator base itself cannot confer efficient aminoacylation to the t RNATrp molecule. Thus, these three base pairs (G2·C71, G3·C70, and G4·C69) in the B. subtilis t RNATrp 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 Trp Rs included G73, G2·C71, G3·C70, and G4·C69.