• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Chemistry: A European journal >Direct monitoring of initiation factor dynamics through formation of 30S and 70S translation-initiation complexes on a quartz crystal microbalance
【24h】

Direct monitoring of initiation factor dynamics through formation of 30S and 70S translation-initiation complexes on a quartz crystal microbalance

机译：通过在石英晶体微量天平上形成30S和70S翻译起始复合物来直接监测起始因子动力学

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA~(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.
机译：翻译起始是一个动态而复杂的过程，需要构建一个由核糖体，mRNA和起始tRNA组成的70S起始复合物（70S-IC）。在70S-IC的形成过程中，起始因子（IF：IF1，IF2和IF3）与核糖体相互作用形成30S起始复合物（30S-IC）和70S-IC。尽管已经进行了一些光谱分析，但是IF的结合和解离的机理仍然不清楚。在这里，我们采用了一个27 MHz的石英晶体微天平（QCM），通过跟踪频率变化（质量变化）来评估翻译起始中细菌IC形成的过程。 N-末端与生物素羧基载体蛋白（bio-BCCP）融合的IF（IF1，IF2和IF3）固定在中性生物素覆盖的QCM板上。通过使用固定在QCM上的bio-BCCP-IF2，可以实时观察到核糖体起始复合物形成的三个步骤，即简单的实时质量变化：将30S复合物与固定的IF2结合，将50S募集到30S -IC，并形成70S-IC。动力学参数暗示IF2从70S-IC的释放可能是翻译起始中的限速步骤。固定在IF3上的QCM表明，IF3对30S复合物的亲和力随着mRNA和fMet-tRNA〜（fMet）的添加而降低，但并未导致与30S-IC的完全解离。这些结果表明IF3结合并保持与IC的结合，并且其相互作用模式在30S-IC和70S-IC的形成过程中发生了改变，并最终被50S结合诱导从IC上解离。本文展示的这种方法学适用于研究IF在其他途径驱动的翻译起始中的作用。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号