Analysis of translocation of tRNA molecules through ribosome using electron microscopy density map and molecular dynamics simulations

Ishida, Hisashi

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. However, the dynamic mechanism of translocation is unclear at the atomic level. We developed an EM density-fitting refinement method using all-atom molecular dynamics simulations including water molecules while retaining the condition that the constituent biomolecules fit in the EM density map. This method was applied to construct sequential states during the translocation using the X-ray crystallography structure of 70S ribosome complexed with EF-G corresponding to the post-translocational state (PDB code: 2WRJ) and two cryo-EM density maps corresponding to the translocational states (EMID code: 1365 and 1363). It was shown that multistep structural changes, such as ratchet-like motion between the small and large ribosomal subunits and head-rotation of the small ribosomal subunit, were observed during the translocation.