Atomic insights reveal fidelity mechanisms of eukaryotic protein synthesis

Abstract

Protein synthesis involves a critical step where messenger RNA (mRNA) and transfer RNAs (tRNAs) must move in tandem to advance the mRNA reading frame by one codon. This process, known as translocation, is catalyzed by elongation factor G (EF-G) in prokaryotes and elongation factor 2 (eEF2) in archaea and eukaryotes. While eEF2 not only accelerates translocation but also maintains reading frame fidelity, high-resolution structural insights into eukaryotic translocation have remained limited compared to the extensively studied prokaryotic system. In our recently published study, we employed cryogenic-electron microscopy (cryo-EM) to determine ten high-resolution reconstructions of the elongating eukaryotic ribosome in complex with the full translocation module, including mRNA, peptidyl-tRNA, and deacylated tRNA (Milicevic et al.,2024). Seven of these structures included ribosome-bound, naturally modified eEF2. These snapshots captured the stepwise progression of the mRNA-tRNA2-peptide module through the eukaryotic 80S ribosome, from the initial accommodation of eEF2 until the final stages of translocation (Milicevic et al.,2024). We further showed a complex network of interactions that safeguards against reading frame slippage during translation. Additionally, we illustrated how the accuracy of translocation in eukaryotes is reinforced by specific features of the 80S ribosome and eEF2. Finally, we suggested that diphthamide, a conserved post-translational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon pairing, but also restricts Wobble geometry of the second base pair.

Keywords: Accuracy; Elongation; Eukaryote; Protein; Ribosome; Translocation; mRNA.