Conformational Changes of the Small Ribosomal Subunit During Elongation Factor G-dependent tRNA–mRNA Translocation

Abstract

Translocation, a coordinated movement of two tRNAs together with mRNA on the ribosome, is catalyzed by elongation factor G (EF-G). The reaction is accompanied by conformational rearrangements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearrangements by restricting the conformational flexibility of the ribosome by antibiotics binding to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hygromycin B (HygB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, including the unlocking rearrangement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. binding of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicating that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA binding in the A site, whereas Spc and HygB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, although it caused a large increase in tRNA affinity to the A site. These results suggest that conformational changes in the vicinity of the decoding region at the binding sites of Spc and HygB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensating the effect on tRNA affinity.