Direct visualization of translational GTPase factor-pool formed around the archaeal ribosomal P-stalk by high-speed atomic force microscopy

The ribosomal stalk protein plays an essential role in the recruitment of translational GTPase factors EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient 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 stalk (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. Intriguingly, archaeal aEF1A and aEF2 molecules spontaneously assembled around the ribosomal P-stalk up to the maximum number of available binding sites. These results provide the first visual evidence for the factor-pooling mechanism and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.