Expression and purification of threonyl tRNA synthetase RNA binding domainfor heteronuclear NMR studies

Heteronuclear NMR is a straightforward technique for the analysis of contac t areas between one protein and its substrates. We have chosen this approac h to study the interaction of threonyl-tRNA synthetase (ThrRS) with its two substrates. ThrRS forms a complex with the anticodon loop of (Thr)tRNA and also with a part of its mRNA, the second interaction being involved in the regulation of ThrRS expression. In these two cases, the interacting part o f ThrRS is mostly limited to its C-terminal domain. A two-step study has be en conducted: using a first genetic construction, we have validated our app roach, which was then modified to improve the solubility and the stability of the recombinant domain. The latter construct was used to prepare an 15N labelled sample which gave heteronuclear NMR spectra of sufficient quality for structure and interaction studies. (C) 2001 Academie des sciences/Editi ons scientifiques et medicales Elsevier SAS.