Brunel C, Romby P, Moine H, Caillet J, Grunberg-Manago M, Springer M, Ehresmann B, Ehresmann C
UPR no 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Biochimie. 1993;75(12):1167-79. doi: 10.1016/0300-9084(93)90016-l.
Previous work showed that E coli threonyl-tRNA synthetase (ThrRS) binds to the leader region of its own mRNA and represses its translation by blocking ribosome binding. The operator consists of four distinct domains, one of them (domain 2) sharing structural analogies with the anticodon arm of the E coli tRNA(Thr). The regulation specificity can be switched by using tRNA identity rules, suggesting that the operator could be recognized by ThrRS as a tRNA-like structure. In the present paper, we investigated the relative contribution of the four domains to the regulation process by using deletions and point mutations. This was achieved by testing the effects of the mutations on RNA conformation (by probing experiments), on ThrRS recognition (by footprinting experiments and measure of the competition with tRNA(Thr) for aminoacylation), on ribosome binding and ribosome/ThrRS competition (by toeprinting experiments). It turns out that: i) the four domains are structurally and functionally independent; ii) domain 2 is essential for regulation and contains the major structural determinants for ThrRS binding; iii) domain 4 is involved in control and ThrRS recognition, but to a lesser 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 contains the ribosome loading site, is not involved in ThrRS recognition. The binding of ThrRS probably masks the ribosome binding site by steric hindrance and not by direct contacts. This is only achieved when ThrRS 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 recognition. It should serve as an articulation to provide an appropriate spacing between domains 2 and 4. Furthermore, it is possibly involved in ribosome binding.
先前的研究表明，大肠杆菌苏氨酰 - tRNA合成酶（ThrRS）与其自身mRNA的前导区域结合，并通过阻止核糖体结合来抑制其翻译。操纵子由四个不同的结构域组成，其中一个结构域（结构域2）与大肠杆菌tRNA（Thr）的反密码子臂具有结构相似性。通过使用tRNA识别规则可以切换调节特异性，这表明操纵子可以被ThrRS识别为类似tRNA的结构。在本文中，我们通过使用缺失和点突变研究了这四个结构域对调节过程的相对贡献。这是通过测试突变对RNA构象（通过探针实验）、对ThrRS识别（通过足迹实验以及测量与tRNA（Thr）在氨酰化反应中的竞争）、对核糖体结合以及核糖体/ThrRS竞争（通过引物延伸实验）的影响来实现的。结果表明：i）这四个结构域在结构和功能上是独立的；ii）结构域2对于调节至关重要，并且包含ThrRS结合的主要结构决定因素；iii）结构域4参与调控和ThrRS识别，但程度低于结构域2。然而，先前描述的与受体样茎的相似性在功能上并不显著。它如何被ThrRS识别仍有待解决；iv）包含核糖体装载位点的结构域1不参与ThrRS识别。ThrRS的结合可能通过空间位阻掩盖核糖体结合位点，而不是通过直接接触。只有当ThrRS与结构域2和4都相互作用时才能实现这一点；v）连接结构域2和4的未配对结构域3不直接参与ThrRS识别。它应作为一个关节，在结构域2和4之间提供适当的间距。此外，它可能参与核糖体结合。
