Two essential regions for tRNA recognition in Bacillus subtilis tryptophanyl-tRNA synthetase.
Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) is a homodimeric enzyme. A model for its ability to recognize tRNA(Trp) in B. subtilis was proposed by using computer modelling. This was based on the the fact that there is high homology among bacterial TrpRSs [Chen, Jiang, Jin and Wang (2001) Acta Biochim. Biophys. Sinica 33, 687-690], in which the enzyme dimer binds to two tRNA(Trp) molecules and each tRNA(Trp) is bound to two different domains across the surface of the dimer. In this work, three deletion mutants of TrpRS were constructed and their products were purified. After determining the kinetic parameters of the mutants in the two-step reaction, it was found that the relative activities of wild-type and mutant enzymes had changed little in the ATP-pyrophosphate exchange reaction. In contrast, the activities of three mutant proteins were much decreased in the tRNA(Trp) aminoacylation assay. Deletion of residues 108-122 and residues 234-238 caused 44% and 80% reductions in the activity, respectively. When both regions were deleted, the aminoacylation activity of the TrpRS mutant was too low to be determined using tRNA(Trp) at the limiting concentration. Gel-retardation assays showed that the acceptor minihelix and the anticodon microhelix were recognized by the domains of TrpRS spanning residues 108-122 and residues 234-238 respectively. In addition, the deletion of amino acids 234-238 affected the normal induced expression of TrpRS at 37 degrees C. In conclusion, residues 108-122 and 234-238 were found essential for tRNA(Trp) recognition.
Jia
Jie
J
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Xu
Feng
F
Chen
Xianglong
X
Chen
Li
L
Jin
Youxin
Y
Wang
Debao T P
DT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000378:metabolism*; D016415:Sequence Alignment; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*
D012329:RNA, Bacterial; D012364:RNA, Transfer, Trp; D000255:Adenosine Triphosphate; D014369:Tryptophan-tRNA Ligase