Abstract
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)(3) (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.