Low Energy Barriers and a Dynamic Contact Network between Ribosomal Subunits Enable Rapid tRNA Translocation

Abstract

During protein synthesis, tRNA molecules move from the ribosome's aminoacyl to peptidyl to exit sites, with the two ribosomal subunits remaining associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. Using molecular dynamics simulations, we here investigate conformational motions during spontaneous translocation, as well as the underlying energetics and kinetics. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and post-translocation states. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by ‘sliding’ and ‘stepping’ mechanisms involving conserved L6, L5, and L1 residues, thus ensuring binding to the ribosome despite large-scale tRNA movement through maintaining constant binding affinity. Intersubunit rotations exhibit remarkably fast intrinsic submicrosecond dynamics, which requires a fine-tuned flat free energy landscape, as any larger barrier would slow down the conformational motions. Maintaining such subtle balance between the many interactions involved is remarkable, in particular considering the large shifts the many intersubunit bridges undergo. Based on the observed occupancies of intersubunit contacts during our simulations, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The total contribution of the tRNAs -- which contact both subunits -- to the intersubunit binding enthalpy is almost constant, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation.