| Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was expressed and purified by the aid of the high-level expression vector pKSW1. The overexpression vector pET-24a(+)-HTrpRS was constructed and human TrpRS was expressed in E.coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His6 tagged human tryptophanyl-tRNA synthetase. A series of tRNATrp variants were prepared by in vitro transcription and their efficiencies of aminoacylation by tryptophan (kcat/KM) were measured with the aid of B.subtilis and human TrpRS. The identity elements in the operational RNA code of human tRNATrp were found to be, major element, discriminator base A73; minor elements, G1/C72 and U5/G68. From the cross-species aminoacylation assays, we conclude that the identity elements in tRNATrps from B.subtilis and human all contribute to the species-specific aminoacylation by TrpRS. Analyses of TrpRS and tRNATrp acceptor stem sequences covering three taxonomic domains (bacteria, eucarya and archaea) reflect coevolution of TrpRS and tRNATrp acceptor stem.A library containing 20-nt random region and TrpRS from B. subtilis were used for in vitro selection. The aptamers share a common structure of three G:C pairs. A series of tRNATrp variants was prepared and assayed by TrpRS from B.subtilis and human. The mutants that possess the three G:C pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B.subtilis tRNATrp, while the G73 discriminator base itself can not confer efficient aminoacylation to tRNATrp molecule. Thus, these three G:C pairs (G2:C71, G3:C70 and G4:C69) in B.subtilis tRNATrp acceptor stem were established to be important recognition elements. Uv-crosslinking reaction was performed and the results indicated that the G73 and T72 base of minihelix DNA interacted TrpRS directly. The uv-crosslinking reaction was improved by the dose of uv irradiation and the concentration of both TrpRS and minihelix DNA.
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