Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation.
Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution.
Vincent
C
C
European Molecular Biology Laboratory, Grenoble, France.
Borel
F
F
Willison
J C
JC
Leberman
R
R
Härtlein
M
M
D000215:Acylation; D000586:Amination; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012362:RNA, Transfer, Ser / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012714:Serine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012362:RNA, Transfer, Ser; D012714:Serine-tRNA Ligase