Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.
Nucleic Acids Res. 2010 Jan;38(2):548-58. doi: 10.1093/nar/gkp908. Epub 2009 Nov 11.
Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.
真核生物的翻译终止是由 eRF1 和 eRF3 因子协同作用控制的。eRF1 识别核糖体 A 位上的终止密码子，并促进新生肽链的释放，GTP 酶 eRF3 通过与 eRF1 的相互作用促进这种肽链释放。除了在终止中的作用外，eRF3 通过其与细胞质多聚（A）结合蛋白（PABP）的结合，通过 eRF3 N 端结构域中的 PAM2-1 和 PAM2-2 基序参与正常和无义介导的 mRNA 衰减。我们使用等温滴定量热法研究了全长 eRF3 与其配体（GDP、GTP、eRF1 和 PABP）之间的复合物形成，证明了 eRF1:eRF3:PABP:GTP 复合物的形成。对 eRF3 与 G 核苷酸相互作用的温度依赖性分析表明，伴随 eRF1:eRF3:GTP 复合物形成发生了主要的结构重排。这与未检测到的 eRF1:eRF3:GDP 复合物形成形成对比。因此，我们的结果与 GTP 在促进翻译终止中的积极作用一致。通过对 eRF3 的 PAM2-1 和 PAM2-2 基序进行定点突变，我们证明 PAM2-2 但不是 PAM2-1 对于 eRF3:PABP 复合物的形成是必不可少的。
