Kinetics of Spontaneous and EF-G-Accelerated Rotation of Ribosomal Subunits

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.

Dynamic relationships between ribosomal conformational and RNA positional changes during ribosomal translocation

“…These results are also consistent with the ensemble kinetic data of Sharma et al [37] showing that for the case of EF-G-induced translocation at saturating EF-G.GTP the time course of S6-L9 FRET has a small downward phase, with a rate constant of k down = 200 or 210 ${\text{s}}^{-1}$, which is followed by an upward phase, with a rate constant of k up = 15 or 11 ${\text{s}}^{-1}$. Thus, our results imply that the experimental data of k down = 200 or 210 ${\text{s}}^{-1}$ in Sharma et al [37] does not necessarily reflect the rate constant of the transition from the classical non-rotated pretranslocation to rotated hybrid state. Consider that a small fraction (denoted by Q ) of the pretranslocation ribosomal complexes are in the classical non-rotated state.…”

“…1 (with values of rate constants k 2 = 180 ${\text{s}}^{-1}$, k 4 = 26 ${\text{s}}^{-1}$ and other rate constants as given in Table 1). Consequently, our theoretical results are consistent with both the smFRET data of Cornish et al [3] showing that EF-G binding only mildly facilitates the transition from the classical non-rotated pretranslocation to rotated hybrid state and the recent ensemble kinetic data of Sharma et al [37] showing a very fast (about 200 ${\text{s}}^{-1}$) decrease of S6-L9 FRET data upon EF-G.GTP binding.…”

“…1 it is noted that at saturating concentration of EF-G.GTP, the slight decrease of the S6-L9 FRET (corresponding to the transition from State H to State H1) proceeds with a rate constant of k decrease = k 2 = 250 ${\text{s}}^{-1}$, which is followed by a large increase of S6-L9 FRET (corresponding to the transition from State H1 through State POST) with a rate constant of k increase = $true(1true/k_3+1true/k_4+1true/k_{5}true)$ = 21 ${\text{s}}^{-1}$. These results are also consistent with the ensemble kinetic data of Sharma et al [37] showing that for the case of EF-G-induced translocation at saturating EF-G.GTP the time course of S6-L9 FRET has a small downward phase, with a rate constant of k down = 200 or 210 ${\text{s}}^{-1}$, which is followed by an upward phase, with a rate constant of k up = 15 or 11 ${\text{s}}^{-1}$. Thus, our results imply that the experimental data of k down = 200 or 210 ${\text{s}}^{-1}$ in Sharma et al [37] does not necessarily reflect the rate constant of the transition from the classical non-rotated pretranslocation to rotated hybrid state.…”

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

“…Recent data indicate that the most rate-limiting step during translocation is the movement of the tRNA anticodon ends, associated with swivelling of the SSU head (26,44,45). Elongation factor 2 facilitates the movement in two ways.…”

Molecular machines

“…The ribosome is a protein machine that makes other protein machines and is arguably the most sophisticated molecular device in the cellular universe. Sarah Adio tracks its mechanical cycle at the single-residue scale (Sharma et al 2016). Living cells are societies of molecular machines.…”