Structure-Based Energetics of Stop Codon Recognition by Eukaryotic Release Factor.

In translation termination, the eukaryotic release factor (eRF1) recognizes mRNA stop codons (UAA, UAG, or UGA) in a ribosomal A site and triggers release of the nascent polypeptide chain from P-site tRNA. eRF1 is highly selective for U in the first position and a combination of purines (except two consecutive guanines, i.e., GG) in the second and third positions. Eukaryotes decode all three stop codons with a single release factor eRF1, instead of two (RF1 and RF2), in bacteria. Furthermore, unlike bacterial RF1/RF2, eRF1 stabilizes the compact U-turn mRNA configuration in the ribosomal A site by accommodating four nucleotides instead of three. Despite the available cryo-EM structures (resolution ∼3.5-3.8 Å), the energetic principle for eRF1 selectivity toward a stop codon remains a fundamentally unsolved problem. Using cryo-EM structures of eukaryotic translation termination complexes as templates, we carried out molecular dynamics free energy simulations of cognate and near-cognate complexes to quantitatively address the energetics of stop codon recognition by eRF1. Our results suggest that eRF1 has a higher discriminatory power against sense codons, compared to that reported earlier for RF1/RF2. The compact mRNA formed specific intra-mRNA interactions, which itself contributed to stop codon specificity. Furthermore, the specificity is enhanced by the loss of protein-mRNA interactions and, most importantly, by desolvation of the incorrect codons in the near-cognate complexes. Our work provides a clue to how eRF1 discriminates between cognate and near-cognate codons during protein synthesis.