Mechanisms for Efficient TRNA Translocation through the Ribosome

Abstract

After peptide bond formation the transfer RNAs (tRNAs) bound to the ribosome translocate by more than 7 nm to adjacent binding sites, accompanied by large-scale conformational motions of the ribosome. Combining cryo-EM reconstructions of translocation intermediates (Fischer, Nature 2010) with high resolution crystal structures, we obtained 13 near-atomic resolution structures. The quality of these structures was validated using recent crystal structures and subsequently all-atom molecular dynamics simulations of the fully solvated 70S ribosome were carried out for each of the 13 intermediate states, totaling 1.5 µs. The obtained dynamics within the intermediate states allowed us to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. These rates revealed rapid motions of the L1-stalk and the small subunit on sub-microsecond timescales, whereas the tRNA motions were seen to be rate-limiting for most transitions. By calculating the free energy of interaction between L1-stalk and tRNA, we obtained molecular forces revealing that the L1-stalk is actively pulling the tRNA from P to E site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by ‘sliding’ and ‘stepping’ mechanisms involving key protein-tRNA contacts, explaining how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements.