SERYL-TRANSFER-RNA SYNTHETASE FROM ESCHERICHIA-COLI - FUNCTIONAL EVIDENCE FOR CROSS-DIMER TRANSFER-RNA BINDING DURING AMINOACYLATION

Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase, Each subunit is composed of two distinc t domains: the N-terminal domain is a 60 Angstrom long, arm-like coile d coil structure built up of two antiparallel alpha-helices, whereas t he C-terminal domain, the catalytic core, is an alpha-beta structure o verlying a seven-stranded antiparallel beta-sheet, Deletion of the arm -like domain (SerRS Delta 35-97) does not affect the amino acid activa tion step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude, In the present study, it was shown th at the formation of heterodimers from two aminoacylation defective hom odimers, the N-terminal deletion and an active site mutant (SerRS E355 Q), restored charging activity, The aminoacylation activity in a mixtu re containing the heterodimers was compared to that of solutions conta ining the same concentrations of homodimer, The activity of the mixtur e was eight times higher than the activities of the homodimer solution s, 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 het erodimer contains only one active site, These results are in full agre ement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer bind ing of tRNA in solution.