Taylor Derek J, Nilsson Jakob, Merrill A Rod, Andersen Gregers Rom, Nissen Poul, Frank Joachim
Howard Hughes Medical Institute, Health Research Inc.,Wadsworth Center, Albany, NY 12201-0509, USA.
EMBO J. 2007 May 2;26(9):2421-31. doi: 10.1038/sj.emboj.7601677. Epub 2007 Apr 19.
On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.
基于核糖体蛋白质合成的动力学数据，mRNA - tRNA复合物转位的机械能被认为是由延伸因子（真核生物中的eEF2，细菌中的EF - G）的GTP水解提供的。我们获得了与ADP - 核糖基化的eEF2（ADPR - eEF2）结合的真核核糖体在GTP水解前后的冷冻电镜重建结果，为分析转位的GTP酶偶联机制提供了结构基础。利用ADP - 核糖基团作为独特的标记，我们观察到ADPR - eEF2的构象变化严格归因于GTP水解。这些运动可能代表了生理环境中天然eEF2的运动，并且足以在转位过程中将mRNA - tRNA复合物与核糖体解码中心的两个普遍保守碱基（大肠杆菌中的A1492和A1493）解偶联。对这些数据的解释提供了一个详细的两步转位模型，该模型始于eEF2 / EF - G结合诱导的小核糖体亚基的棘轮运动。然后GTP水解使mRNA - tRNA复合物与解码中心解偶联，这样mRNA - tRNA部分的转位可能通过小亚基的头部旋转来完成。
