多通道smFRET研究揭示了EF-G在核糖体上的紧形状
Jordan L Johnson1, Jacob H Steele1, Ran Lin1
1Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA.
bioRxiv : the preprint server for biology
|February 8, 2024
概括
延长因子G (EF-G) 在核糖体转位过程中经历了构造变化. 模仿人类eEF2修饰的突变将EF-G陷入紧状态,抑制蛋白质合成.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 结构生物学 结构生物学
背景情况:
- 延长因子G (EF-G) 对于细菌核糖体转移至关重要.
- 在EF-G功能中GTP水解的确切作用尚未完全理解.
- 在Thr56中对人类真核延长因子2 (eEF2) 的酸化抑制了蛋白质合成,但机制尚不清楚.
研究的目的:
- 通过单分子Förster共振能量转移 (smFRET) 来研究大肠杆菌EF-G的结构动力学.
- 探索模仿eEF2酸化的突变对EF-G功能和构造的影响.
- 阐明EF-G介导的核糖体转位及其调节的机制.
主要方法:
- 开发一种多通道smFRET显微镜技术.
- 使用双标记的EF-G (Alexa 488/594) 和与核糖体结合的tRNA (Cy3) 和核糖体蛋白L27 (Cy5).
- 分析了EF-G在被标记的核糖体内fMet-Phe-tRNA(Phe) 转位期间的构造变化.
主要成果:
- 野生类型的EF-G在与GTP或GDPCP结合后采用扩展形状,促进转移.
- 突变的EF-G变体 (T48E,T48V) 保留了GTP结合和水解,但表现出受损的Poly () 合成.
- 这些突变物采用了紧的形状,与与sarcin/ricin循环单独的相互作用不同,阻碍了转位.
结论:
- EF-G表现出对其在核糖体转移中的功能至关重要的构造性适应性.
- 该研究提供了关于eEF2修饰如何影响蛋白质合成的机制性见解.
- 这些发现凸显了EF-G特定形状对于高效的蛋白质合成的重要性.
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