Ribosomal Protein L7/L12 is Required for GTPase Translation Factors EF-G, RF3 and IF2 to Bind in their GTP State to 70S Ribosomes

Ribosomal protein L7/L12 is associated with translation initiation, elongation and termination by the 70S ribosome. The GTPase activity of EF-G requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from E. coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2 and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3 and IF2 decreased to basal levels while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3 and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained >50% binding. Lastly, an EF-GΔG′ truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 CTD, either through a G′ domain (EF-G, RF3) or a unique NTD (IF2). Further, we hypothesize this interaction is concomitant with counter-clockwise ribosomal intersubunit rotation, which is required for translocation, initiation and post-termination.