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Ribosomal protein L7/L12 is required for GTP ase translation factors EF ‐G, RF 3, and IF 2 to bind in their GTP state to 70S ribosomes

2017; Wiley; Volume: 284; Issue: 11 Linguagem: Inglês

10.1111/febs.14067

1742-4658

Markus A. Carlson, Bassam G. Haddad, Amanda J. Weis, Colby S. Blackwood, Catherine Shelton, Michelle E. Wuerth, Justin D. Walter, Paul Spiegel,

RNA Research and Splicing

Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5′ triphosphate hydrolase ( GTP ase) activity of elongation factor G ( 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 Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTP ase factors EF ‐G, RF 3, IF 2, and LepA. Upon removal of L7/L12 from ribosomes, the GTP ase activities of EF ‐G, RF 3, and IF 2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTP ase activities of all GTP ases returned to full activity. Moreover, ribosome binding assays indicated that EF ‐G, RF 3, and IF 2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF ‐G∆G′ truncation mutant possessed ribosome‐dependent GTP ase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome‐dependent GTP ases that harbor direct interactions to the L7/L12 C‐terminal domains, either through a G′ domain ( EF ‐G, RF 3) or a unique N‐terminal domain ( IF 2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post‐termination.