Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

Significance Elongation factor Tu (EF-Tu) facilitates rapid and accurate selection of aminoacyl-tRNA (aa-tRNA) by the bacterial ribosome during protein synthesis. We show that EF-Tu dissociates from the ribosome as aa-tRNA navigates the accommodation corridor en route to peptide bond formation. We find that EF-Tu’s release from the ribosome during aa-tRNA selection can be reversible. We also demonstrate that new ternary complex formation, accompanied by futile cycles of GTP hydrolysis, can occur on aa-tRNA bound within the ribosome. These findings inform on the decoding mechanism, the contributions of EF-Tu to the fidelity of translation, and the potential consequences of reduced rates of peptide bond formation on cellular physiology. The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

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