Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis.
Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp). It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp). Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp). These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp). The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp).
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031, China.
Jie
J
Jia
Xiang-Long
XL
Chen
Li-Tao
LT
Guo
Ya-Dong
YD
Yu
Jian-Ping
JP
Ding
You-Xin
YX
Jin
D000215:Acylation; D001412:Bacillus subtilis / Q000235:genetics*; D018698:Glutamic Acid; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D012329:RNA, Bacterial / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000378:metabolism*; D016415:Sequence Alignment; D013045:Species Specificity; D014369:Tryptophan-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D012329:RNA, Bacterial; D012364:RNA, Transfer, Trp; D018698:Glutamic Acid; D014369:Tryptophan-tRNA Ligase; D008239:Lysine