Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, 920-1192 Kanazawa, Japan;
The Institute of Science and Technology, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
Proc Natl Acad Sci U S A. 2020 Dec 22;117(51):32386-32394. doi: 10.1073/pnas.2018975117. Epub 2020 Dec 7.
In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1) (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.
在翻译延伸过程中，两个翻译延伸因子 EF1A 和 EF2 交替结合核糖体并促进多肽延伸。核糖体柄是一个多聚核糖体蛋白复合物，在 EF1A 和 EF2 与核糖体的招募及其 GTP 水解方面发挥着至关重要的作用，从而实现高效和准确的翻译延伸。然而，由于核糖体柄的柔性性质，其结构动力学和作用机制仍不清楚。在这里，我们应用高速原子力显微镜（HS-AFM）直接观察古菌七聚体核糖体柄复合物 aP0•(aP1•aP1)（P 柄）的作用。HS-AFM 电影清楚地表明 P 柄在核糖体大亚基上的摆动运动，其中柄基部采用两种构象状态，一种是预测的典型状态，另一种是新鉴定的翻转状态。此外，我们表明，多达七个古菌 EF1A（aEF1A）和古菌 EF2（aEF2）组装在核糖体 P 柄周围，对应于 P 柄的常见 C 端因子结合位点的拷贝数。这些结果为 P 柄在核糖体内的因子聚集机制提供了直观的证据，并揭示了核糖体 P 柄通过增加翻译延伸因子的局部浓度来促进翻译延伸。
