Species-specific Recognition Of TRNA By The Class Ic Aminoacyl-tRNA Synthetase

Three deletion mutants of Bacillus subtilis TrpRS was constructed, purified and characterized. In the tRNATrp amoinoacylation assay, deletion of the residues 108-122 and the residues 234-238 caused 44% and 80% reductions in the activity, respectively. Gel-retardation assay showed that the acceptor minihelix and the anticodon microhelix could not be recognized when the domains of TrpRS spanning the 108-122 residues and the 234-238 residues were deleted, respectively. In conclusion, the residues 108-122 and 234-238 were found essential for tRNATrp recognition.Conservative residues K149 and E153, which were located beside the pocket of Trp, showed the phylogenic diversities from prokaryote to eukaryote. It should be emphasized E153G has no detectable activity in the activation of Trp until the tRNATrp involves in. In addition, we successfully switched the species-specificity of Bacillus subtilis TrpRS in the recognition of tRNATrp. And WBHA and E153K preferred indeed human tRNATrp to its cognate prokaryotic tRNATrp. We did the homologous analysis in all of the known Class Ic aminoacyl-tRNA synthetases and their cognate tRNA genes. Two conservative resides that located beside the pocket of amino acid substrate was identified with the phylogenetic diversities from prokaryote to eukaryote. And these conservative residues in the AARS might coevolve with the identity elements in its cognate tRNA. The kinetics parameters of the TrpRS mutants disclosed that the charges of the conservative residues showed preference for the discriminator and the first base-pair in the tRNATrp. When the charge of the residue was switched, the species-specificity of TrpRS was switched together. It was identified that in the Class Ic aminoacyl-tRNA synthetases there were the species-specific elements, which charged for the recognition of tRNATrp and extended the genetic code. In addition to, we proposed the potential peptides for tRNA recognition in the TyrRS and TrpRS from B. subtilis and H. sapiens. We thought the acceptor stem of tRNA bond to a groove that consisted of two α helices and identified the identity elements in the tRNA. The His6-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95D. It was confirmed by sequencing and was cloned into the expression vector pET-24a (+) to yield pET-24a (+)-HTyrRS, which was transfected into E.coli BL21- CodonPlus-RIL. The induced-expression level of His6-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni2+) chelate affinity chromatography. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.