Abstract
Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.