Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.
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.
['Imai|Hirotatsu|H|0000-0003-3927-1660', 'Uchiumi|Toshio|T|0000-0002-0001-4344', 'Kodera|Noriyuki|N|0000-0003-4880-8423']
D019843:Archaeal Proteins D004926:Escherichia coli D020963:GTP Phosphohydrolase-Linked Elongation Factors D020558:GTP Phosphohydrolases D006153:Guanosine Diphosphate D006160:Guanosine Triphosphate D018625:Microscopy, Atomic Force D046912:Multiprotein Complexes D010441:Peptide Chain Elongation, Translational D043902:Pyrococcus horikoshii D012269:Ribosomal Proteins D054658:Ribosome Subunits, Large
ribosomal P-stalk ribosome translation elongation trGTPase HS-AFM