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Abstract

Ribosome dynamics play an important role in translation. The rotation of the ribosomal subunits relative to one another is essential for tRNA-mRNA translocation. An important unresolved question is whether subunit rotation limits the rate of translocation. Here, we monitor subunit rotation relative to peptide bond formation and translocation using ensemble kinetics and single-molecule FRET. We observe that spontaneous forward subunit rotation occurs at a rate of 40 s-1, independent of the rate of preceding peptide bond formation. Elongation factor G (EF-G) accelerates forward subunit rotation to 200 s-1. tRNA-mRNA movement is much slower (10-40 s-1), suggesting that forward subunit rotation does not limit the rate of translocation. The transition back to the non-rotated state of the ribosome kinetically coincides with tRNA-mRNA movement. Thus, large-scale movements of the ribosome are intrinsically rapid and gated by its ligands such as EF-G and tRNA.