Abstract
Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. This is achieved by specific release factor proteins that are very different in bacteria and eukaryotes. Hence, while there are two release factors with overlapping specificity in bacteria, the single omnipotent eRF1 release factor in eukaryotes is able to read all three stop codons. This is particularly remarkable as it is able to select three out of four combinations of purine bases in the last two codon positions. With recently determined 3D structures of eukaryotic termination complexes, it has become possible to explore the origin of eRF1 specificity by computer simulations. Here, we report molecular dynamics free energy calculations on these termination complexes, where relative eRF1 binding free energies to different cognate and near-cognate codons are evaluated. The simulations show a high and uniform discrimination against the near-cognate codons, that differ from the cognate ones by a single nucleotide, and reveal the structural mechanisms behind the precise decoding by eRF1.
Highlights
Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity
The structures further show that the mRNA stop codon and the N-domain of eRF1, which is responsible for codon recognition, maintain the same overall conformation in the decoding site throughout the termination process
To evaluate the relative free energies of binding to different codons single-nucleotide mutations were performed, starting from each of the different stop codons. These calculations were carried out both with and without eRF1 bound to the ribosome, which allows the relative binding free energies to be evaluated by a standard thermodynamic cycle[29]
Summary
Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. The omnipotent RF can recognize three combinations of purines in the second and third codon position (UAA, UAG, UGA), but avoid reading the fourth alternative (UGG) (Fig. 1) How this can be achieved from a structural viewpoint has remained a mystery, but recent cryo-EM structures[3,4] of eRF1 complexes with the ribosome hint at a solution of the problem. The latest cryo-EM structures[5] reveal that eRF1 first binds in its closed conformation, similar to what has been suggested for bacterial RFs6,7 This is followed by GTP hydrolysis and dissociation of eRF3, whereupon eRF1 accommodates on the ribosome and inserts the universally conserved GGQ motif of its M domain into the A-site of the peptidyltransferase center (PTC)[5,8]. Paramecium tetraurelia is of particular interest here since the highly conserved Glu[55] and Thr[58] residues are mutated to Asn and Glu, respectively, Cys[127] remains invariant
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