TRANSLATIONAL REGULATION OF THE ESCHERICHIA-COLI THREONYL-TRANSFER-RNA SYNTHETASE GENE - STRUCTURAL AND FUNCTIONAL IMPORTANCE OF THE THRS OPERATOR DOMAINS

Previous work showed that E coil threonyl-tRNA synthetase (ThrRS) bind s to the leader region of its own mRNA and represses its translation b y blocking ribosome binding. The operator consists of four distinct do mains, one of them (domain 2) sharing structural analogies with the an ticodon arm of the E coil tRNA(Thr). The regulation specificity can be switched by using tRNA identity rules, suggesting that the operator c ould be recognized by ThrRS as a tRNA-like structure. In the present p aper, we investigated the relative contribution of the four domains to the regulation process by using deletions and point mutations. This w as achieved by testing the effects of the mutations on RNA conformatio n (by probing experiments), on ThrRS recognition (by footprinting expe riments and measure of the competition with tRNA(Thr) for aminoacylati on), on ribosome binding and ribosome/ThrRS competition (by toeprintin g experiments). It turns out that: i) the four domains are structurall y and functionally independent; ii) domain 2 is essential for regulati on and contains the major structural determinants for ThrRS binding; i ii) domain 4 is involved in control and ThrRS recognition, but to a le sser degree than domain 2. However, the previously described analogies with the acceptor-like stem are not functionally significant. How it is recognized by ThrRS remains to be resolved; iv) domain 1, which con tains the ribosome loading site, is not involved in ThrRS rcognition. The binding of ThrRS probably masks the ribosome binding site by steri c hindrance and not by direct contacts. This is only achieved when Thr RS interacts with both domains 2 and 4; and v) the unpaired domain 3, which connects domains 2 and 4, is not directly involved in ThrRS reco gnition. It should serve as an articulation to provide an appropriate spacing between domains 2 and;4. Furthermore, it is possibly involved in ribosome binding.