Disordered C-terminal domain of tyrosyl transfer-RNA synthetase: evidence for a folded state

The C-terminal domain (residues 320 to 419) of tyrosyl-tRNA synthetaseGroupe d’Ingénierie des from Bacillus stearothermophilus (Bst-TyrRS) is necessary for the binding ofProtéines (CNRS URA1129) tRNATyr but disordered in the crystal structure. Four different criteriaUnite ́ de Biochimie Cellulaire Institut Pasteur, 28 rue du showed that the isolated C-terminal domain of Bst-TyrRS was at least Docteur Roux, 75724, Paris partially folded in solution. Its spectrum of circular dichroism was compatible with a high content of secondary structure elements (56 % of itsCedex 15, France residues) and these structural elements disappeared in 7.5 M urea. It was unfolded by urea along a unique transition, around 6.0 M, according to the variations in the fluorescence of its tyrosine residues and in its electrophoretic mobility through transverse gradient gels. It was denatured by heat with a temperature of half-precipitation in 30 minutes that was equal to 67.9°C and close to the Bst-TyrRS one, 68.7°C. Its transitions of denaturation by urea or temperature were weakly cooperative. The C-terminal domains of the TyrRSs from Escherichia coli (Eco-TyrRS) and B. stearothermophilus could be genetically exchanged without a significant loss of aminoacylation activity. A hybrid between the N-terminal domain of Bst-TyrRS and the C-terminal domain of Eco-TyrRS was precipitated by heat in 30 minutes following two transitions: 83 % of the molecules were precipitated with a temperature of half-transition (51.6°C) close to the Eco-TyrRS one (48.6°C). The remainder was precipitated with a temperature of half-transition (65.5°C) close to the Bst-TyrRS one (67.2°C) or that of its N-terminal domain (68.0°C). These results showed that the C-terminal domain of Eco-TyrRS could undergo a transition from a soluble active conformation to an insoluble one. The denaturations of Bst-TyrRS and of its N-terminal domain by urea occurred with two successive transitions, around 4 M and 6 M, and thus according to a complex mechanism