Free-Energy Landscape of Ribosome Translocation Analysed using MD Simulations and Cryo-EM Density Maps

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Ribosome is one of the supra-biomolecules used in the process of translating genetic information for the synthesis of polypeptides. In the course of its synthesis, two tRNA molecules move with mRNA through ribosome, changing their positions at the A (animoacyl), P (peptidyl), and E (exit) sites. This process, called translocation of tRNAs, is catalyzed by the elongation factor G (EF-G) using energy of GTP hydrolysis. Recent results from pre-steady-state kinetic analysis and cryo-electron microscopy (cryo-EM) suggest that there is a dynamic multistep process during translocation, where large-scale structural rearrangements of both ribosome and EF-G occur. However, the dynamic mechanism of translocation is unclear at the atomic level.We used all-atom molecular dynamics (MD) simulations to direct 70S ribosome complexed with EF-G at the post-translocational state (PDB code: 2WRJ) towards the translocational and pre-translocational states (EMDB code: EMD-1365 and EMD-1363) by fitting 70S ribosome into cryo-EM density maps. Additionally, the simulations were assisted by umbrella sampling simulations, in which biased potentials were imposed on the centers of masses of the protein molecules in the 70S ribosome, to relax the transitional conformations and to construct the free-energy landscape of the translocation. Multistep structural changes, such as a ratchet-like motion between the small and large ribosomal subunits, and a hinge-like motion of elongation factor G (EF-G) were observed during the translocation. The free-energy landscape shows that there are semi-stable states between two stable states at the pre- and post-translational states. It was shown that a loop of nucleic acids from the large ribosomal subunit, which is located near the P- and E-sites, plays an important role in the translocation of P-tRNA and E-tRNA.