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Abstract
To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA.
Highlights
The ribosome is a supra-molecule which synthesizes protein by translating genetic information to the amino acid sequence
After codon-anticodon recognition, aminoacyl-tRNA is in the classical A/A state, while peptidyl-tRNA is in the classical P/P state. (As for X/Y, where X, Y = A or P or E, and X represents the binding site of the anticodon stem loops (ASL) of tRNAs on the small ribosomal subunit and Y the site of the acceptor ends of tRNAs on the large ribosomal subunit.) Translocation starts after messenger RNA is decoded and peptide bonds are formed by the peptidyl-transferase reaction
To prepare the initial atomic structure for the fitting, we used the atomic structure of Thermus thermophilus 70S ribosome in the POST state which was determined by X-ray crystallography (PDB code: 2WRI/2WRJ, resolution: 3.6 A ) [10]
Summary
The ribosome is a supra-molecule which synthesizes protein by translating genetic information to the amino acid sequence. MRNA and tRNA move through a solvent-accessible channel located at the interface of the large and small ribosomal subunits (50S and 30S, respectively, in bacteria). (As for X/Y, where X, Y = A or P or E, and X represents the binding site of the anticodon stem loops (ASL) of tRNAs on the small ribosomal subunit and Y the site of the acceptor ends of tRNAs on the large ribosomal subunit.) Translocation starts after messenger RNA (mRNA) is decoded and peptide bonds are formed by the peptidyl-transferase reaction. During the peptidyl-transferase reaction, the growing polypeptide chain is transferred to the A-site tRNA from the P-site tRNA in classical P/P state, resulting in the formation of the pretranslocational (PRE) complex.
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