FEBS Journal, June 2017, Vol.284(11), pp.1631-1643
Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5′ triphosphate hydrolase (ase) activity of elongation factor G (‐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 70S ribosomes to analyze the effect of L7/L12 on the activities of the ase factors ‐G, 3, 2, and LepA. Upon removal of L7/L12 from ribosomes, the ase activities of ‐G, 3, and 2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the ase activities of all ases returned to full activity. Moreover, ribosome binding assays indicated that ‐G, 3, and 2 require L7/L12 for stable binding in the state, and LepA retained 〉 50% binding. Lastly, an ‐G∆G′ truncation mutant possessed ribosome‐dependent ase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome‐dependent ases that harbor direct interactions to the L7/L12 C‐terminal domains, either through a G′ domain (‐G, 3) or a unique N‐terminal domain (2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post‐termination. The ribosomal protein L7/L12 is thought to be important for translation initiation, elongation and termination by the 70S ribosome. However, its precise role in these reactions was unclear. In this study, Spiegel and colleagues develop methods to unambiguously study the importance of L7/L12 in translation. They show that the presence of L7/L12 is required for the binding and activity of GTPases that form direct contacts with L7/L12, including EF‐G, RF3, and IF2. In contrast, the GTPase LepA, which does not make direct contacts with L7/L12, remains active in its absence. These results suggest that L7/L12 is required for stable binding of ribosome‐dependent GTPases that make direct interactions with L7/L12.
Ribosome ; Translation ; Elongation Factor G ; Protein L7/L12 ; Gtp Ase
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