During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.

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Significance The ribosome decodes genetic information and synthesizes proteins in all living organisms. To translate the genetic information, the ribosome binds tRNA. During polypeptide chain elongation, the tRNA is moved together with the mRNA through the ribosome. This movement is called translocation and involves precisely coordinated steps that include binding of a protein called elongation factor G (EF-G). How exactly EF-G drives translocation is not fully understood. We show in this study a detailed three-dimensional molecular image of the ribosome bound to EF-G and two tRNAs, just before the tRNAs are translocated. The image provides mechanistic clues to how EF-G promotes tRNA translocation.

The authors note that on page 2572, left column, in line 16 of the second full paragraph of the Results section, the sentence, ''Based on the dye locations and previous biochemical and structural characterization of similar complexes, we infer that ribosome populations primarily in a low-FRET regime correspond to an open state of the L1 stalk with populations primarily in the high-FRET regime in a closed conformation of the L1 stalk in the P/E hybrid state,'' should instead read ''Based on the dye locations and previous biochemical and structural characterization of similar complexes, we infer that the low-FRET population corresponds to an open state of the L1 stalk and the high-FRET population to a closed conformation of the L1 stalk in the P/E hybrid state.'' The authors note that due to a printer's error, on page 2575, left column, in line 4 of the first full paragraph, the sentence, ''After the movement of the L1 stalk, we were able to enrich for populations of half-closed (Ϸ0.4 FRET) complexes by addition of excess deacylated tRNA to classical state (Ϸ0.25 FRET) complexes containing a vacant E site ( Fig. 3 C and E),'' should instead appear as ''Following the movement of the L1 stalk, we were able to enrich for populations of half-closed (Ϸ0.4 FRET) complexes by addition of excess deacylated tRNA to classical state (Ϸ0.25 FRET) complexes containing a vacant E site ( Fig. 3 C and E).'' Additionally, the authors note that in Fig. 6, the y-axis of panel B was labeled incorrectly. The corrected figure and its legend appear below. (9) and L1 stalk movement (this work; Table S1). The dashed lines at K eq ϭ 1 divide the plot into 4 quadrants corresponding to the 4 possible combinations of nonrotated and rotated orientations of the subunits and fully closed and open conformations of the L1 stalk. Filled squares correspond to complexes of pretranslocation ribosomes containing deacylated tRNA in the P site (tRNA Tyr , tRNA Phe , or tRNA fMet ). Open circles correspond to posttranslocation ribosomes containing N-Ac-Phe-tRNA Phe in the P site and a vacant E site. Open triangles correspond to vacant ribosomes with or without EF-G⅐GDPNP bound. (B) Correlation between forward rates: closing of the L1 stalk vs. rotation of subunits from classical to hybrid state. (C) Correlation between reverse rates: opening of the L1 stalk vs. rotation of subunits from hybrid to classical state. Lines represent log-linear fits of the data.

During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor EF-G. Based on cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We have used a combination of two fluorescence-based approaches to study the timing of these events: Intersubunit FRET measurements to observe relative rotational movement of the subunits and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G·GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. Comparison of the rates of these movements reveals that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state.