Following movement of domain IV of elongation factor G during ribosomal translocation

Significance The ribosome synthesizes proteins in all living organisms. During the process of protein synthesis, tRNAs and mRNA move through the ribosome, and this movement is catalyzed by the binding of a protein called elongation factor G (EF-G). The mechanism of EF-G–induced translocation of mRNA and tRNAs is not fully understood. In this work, we show that EF-G undergoes structural rearrangements and adopts at least two distinct conformations during translocation. Our work provides new insights into how EF-G promotes tRNA and mRNA translocation. Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G–ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.