阐释延伸因子EF4的分子机制和生理功能

延伸因子EF4是细胞内最为保守的蛋白质之一，仅次于延伸因子EF-G和EF-Tu。EF4、EF-G和EF-Tu都有着类似的蛋白结构域和三维结构。EF-G和EF-Tu是翻译过程所绝对必需的辅助因子。但EF4参与翻译过程中的分子机制及其生理功能至今仍不清楚。.近期研究表明，EF4可与核糖体结合，并在某些条件下对蛋白表达水平起着一定的调控作用。在此，我们计划结合先进的单分子荧光技术和新兴的核糖体图谱技术来揭示EF4的机制和功能。一方面，利用基于全内反射荧光显微镜的四色荧光共振能量转移技术，从多个维度细致表征EF4与核糖体相互作用时的结构和动力学信息，包括：EF4自身构型变化、EF4诱导的核糖体构型变化、以及相互作用时两者之间的调控等。另一方面，利用核糖体图谱技术在细胞层面高通量的筛选EF4在翻译过程的功能位点。最后结合两个层面的认知，阐述EF4的分子机制和生理功能及其两者之间的联系。

Besides elongation factor G (EF-G) and elongation factor Tu (EF-Tu), elongation factor 4 (EF4) is the most conserved protein and presents in almost all bacteria. EF4 has similar protein domains and 3D structures as EF-G and EF-Tu. EF-G and EF-Tu are absolute essential protein factors for elongation cycles of translation. However, molecular mechanisms and physiological functions of EF4 in translation remain unknown..Recent studies showed that EF4 interacts with the ribosome in vivo and modulates protein expression level under certain conditions. Here, we propose to combine cutting-edge single molecule fluorescence techniques with newly emerging ribosome profiling method to shed lights on the mechanisms and functions of EF4. On one hand, we will combine total internal reflection fluorescence (TIRF) microscope with four-color fluorescence resonance energy transfer (FRET) to carefully examine interaction between EF4 and the ribosome during translation and to obtain corresponding structural change and kinetic information at molecular level, including conformational changes of EF4, conformational changes of the ribosome induced by EF4 binding, and modulation between them. On the other hand, we will use ribosome profiling method to screen and locate where EF4 modulates translation in cell. By combining knowledge gained from two different methods, we will be able to reveal the molecular mechanisms and physiological functions of EF4 and elucidate how its molecular mechanisms lead to its functions at cellular level.