Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China.
J Microbiol Biotechnol. 2012 Feb;22(2):176-83. doi: 10.4014/jmb.1108.08019.
Eukaryotic translation termination is governed by eRF1 and eRF3. eRF1 recognizes the stop codons and then hydrolyzes peptidyl-tRNA. eRF3, which facilitates the termination process, belongs to the GTPase superfamily. In this study, the effect of the MC domain of eRF1a (eRF1aMC) on the GTPase activity of eRF3 was analyzed using fluorescence spectra and high-performance liquid chromatography. The results indicated eRF1aMC promotes the GTPase activity of eRF3, which is similar to the role of eRF1a. Furthermore, the increased affinity of eRF3 for GTP induced by eRF1aMC was dependent on the concentration of Mg(2+). Changes in the secondary structure of eRF3C after binding GTP/GDP were detected by CD spectroscopy. The results revealed changes of conformation during formation of the eRF3C·GTP complex that were detected in the presence of eRF1a or eRF1aMC. The conformations of the eRF3C·eRF1a·GTP and eRF3C·eRF1aMC·GTP complexes were further altered upon the addition of Mg(2+). By contrast, there was no change in the conformation of GTP bound to free eRF3C or the eRF3C·eRF1aN complex. These results suggest that alterations in the conformation of GTP bound to eRF3 is dependent on eRF1a and Mg(2+), whereas the MC domain of eRF1a is responsible for the change in the conformation of GTP bound to eRF3 in Euplotes octocarinatus.
真核翻译终止由 eRF1 和 eRF3 调控。eRF1 识别终止密码子，然后水解肽酰-tRNA。促进终止过程的 eRF3 属于 GTPase 超家族。在这项研究中，使用荧光光谱和高效液相色谱分析了 eRF1a 的 MC 结构域（eRF1aMC）对 eRF3 GTPase 活性的影响。结果表明，eRF1aMC 促进 eRF3 的 GTPase 活性，这与 eRF1a 的作用相似。此外，eRF1aMC 诱导的 eRF3 对 GTP 的亲和力增加依赖于 Mg2+浓度。通过圆二色性（CD）光谱检测到 eRF3C 结合 GTP/GDP 后二级结构的变化。结果表明，在存在 eRF1a 或 eRF1aMC 的情况下，检测到 eRF3C·GTP 复合物形成过程中构象的变化。在添加 Mg2+后，eRF3C·eRF1a·GTP 和 eRF3C·eRF1aMC·GTP 复合物的构象进一步改变。相比之下，游离 eRF3C 或 eRF3C·eRF1aN 复合物结合的 GTP 的构象没有变化。这些结果表明，eRF3 结合的 GTP 构象的变化依赖于 eRF1a 和 Mg2+，而 eRF1a 的 MC 结构域负责八肋游仆虫中 eRF3 结合的 GTP 构象的变化。
