Johnson Jordan L, Steele Jacob H, Lin Ran, Stepanov Victor G, Gavriliuc Miriam N, Wang Yuhong
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA.
bioRxiv. 2024 Jan 28:2024.01.27.577133. doi: 10.1101/2024.01.27.577133.
While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-G's indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of EF-G induced by mutations that closely aligned with eEF2's Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNA-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which wasn't observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.
虽然延伸因子G（EF-G）对核糖体转位至关重要，但其GTP水解的作用仍不明确。人真核延伸因子2（eEF2）在苏氨酸56位点的磷酸化进一步证明了EF-G的不可或缺性，这种磷酸化会全面抑制蛋白质合成，但其确切机制尚不清楚。在本研究中，我们开发了一种多通道单分子荧光共振能量转移（smFRET）显微镜方法，以研究与eEF2的苏氨酸56残基紧密对齐的突变所诱导的EF-G的构象变化。我们利用Alexa 488/594双标记的EF-G催化甲酰甲硫氨酸-苯丙氨酸- tRNA-Cy3在Cy5-L27标记的核糖体内的转位，使我们能够在同一复合物中探测这两个过程。我们的研究结果表明，在存在GTP或GDP- β - 氯嘌呤（GDPCP）的情况下，野生型EF-G在与核糖体结合时会发生构象延伸，以促进正常转位。另一方面，T48E和T48V突变不影响GTP/GDP结合或GTP水解，但阻碍了多聚（苯丙氨酸）的合成，并使EF-G呈现出一种独特的紧凑构象，当这些突变体仅与帚曲霉素/蓖麻毒素环相互作用时未观察到这种构象。这项研究为EF-G的适应性提供了新的见解，并揭示了人eEF2的修饰机制。
